Hydroxy-Substituted Heteroarylpiperazines: Novel Scaffolds for Î²

Article pubs.acs.org/jmc

Hydroxy-Substituted Heteroarylpiperazines: Novel Scaffolds for β‑Arrestin-Biased D2R Agonists Barbara Man̈ nel, Daniela Dengler, Jeremy Shonberg, Harald Hübner, Dorothee Möller, and Peter Gmeiner* Department of Chemistry and Pharmacy, Medicinal Chemistry, Friedrich-Alexander University Erlangen-Nuernberg, Schuhstraße 19, 91052 Erlangen, Germany S Supporting Information *

ABSTRACT: By means of a formal structural hybridization of the antipsychotic drug aripiprazole and the heterocyclic catecholamine surrogates present in the β2-adrenoceptor agonists procaterol and BI-167107 (4), we designed and synthesized a collection of novel hydroxy-substituted heteroarylpiperazines and heteroarylhomopiperazines with high dopamine D2 receptor (D2R) affinity. In contrast to the weak agonistic behavior of aripiprazole, these ligands are capable of effectively mimicking those interactions of dopamine and the D2R that are crucial for an active state, leading to the recruitment of β-arrestin-2. Interestingly, some ligands show considerably lower intrinsic activity in guanine nucleotide exchange experiments at D2R and consequently represent biased agonists favoring β-arrestin-2 recruitment over canonical G protein activation. The ligands’ agonistic properties are substantially driven by the presence of an endocyclic H-bond donor.

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INTRODUCTION The five different dopamine receptor subtypes (D1R−D5R) are widely distributed throughout the body, especially within the central nervous system (CNS). These G protein-coupled receptors (GPCRs) mediate the physiological functions of dopamine, a neurotransmitter and hormone with a catecholamine structure.1 Not surprisingly, malfunctions of the dopaminergic neurotransmission are involved in a number of pathophysiological conditions including schizophrenia, addiction, and Parkinson’s disease (PD). PD is characterized by severe locomotor deficits, which are mainly caused by the loss of dopaminergic neurons in the substantia nigra pars compacta. Besides the cardinal symptoms akinesia, rigidity, and tremor at rest, patients also exhibit cognitive and other nonmotor symptoms in both advanced and early stages of the disease.2 Despite considerable efforts to develop novel therapeutic agents, L-DOPA (L-3,4-dihydroxyphenylalanine) applied together with a peripheral decarboxylase inhibitor remains the “gold standard” in symptomatic antiparkinsonian therapy. However, the prolonged use of this dopamine precursor results in a loss of efficacy over time, resulting first in “off-phases” and concomitantly occurring L-DOPA-induced dyskinesias (LID) during the “on-phases” at later stages of the disease.3 Attempts to improve fluctuations or to delay the onset of fluctuations and LIDs with D2R agonists as monotherapy or in combination © 2017 American Chemical Society

with a lower L-DOPA dose did not prove successful, as the onset of LID is not dependent on the duration of therapy but rather the duration of the disease.4,5 The exact mechanisms underlying LIDs remain a matter of debate and probably depend on the overall context of denervation and compensatory processes in the CNS. However, animal models of PD suggest LIDs may be associated with uncontrolled neuronal excitability and dopamine receptor supersensitivity.6,7 Activation of D2Rs, as for a plethora of other GPCRs, leads to conformational changes transmitted from the ligand-binding pocket toward intracellular receptor domains. Distinct receptor conformations are thought to guide the intracellular signaling events by interaction of the receptor with different signal transducers, namely G proteins and β-arrestins.8 Using evolutionary trace for the generation of D2R mutants specifically interacting with the G protein or β-arrestin signaling pathway, it was demonstrated that G protein- and β-arrestinmediated signaling at the D2R is dissociable and has distinct physiological outcomes. Although the generated mutant receptors differed in only two residues compared to wildtype, only the β-arrestin-specific D2R mutant was able to promote significant amphetamine-induced forward locomotion Received: March 7, 2017 Published: May 10, 2017 4693

DOI: 10.1021/acs.jmedchem.7b00363 J. Med. Chem. 2017, 60, 4693−4713

Journal of Medicinal Chemistry

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Chart 1. Previously Described β-Arrestin-Biased Agonists 1−3 with Partial Agonist Characteristics at D2R

Chart 2. Formal Structural Hybridization of the D2R Partial Agonist Aripiprazole and the Known β2-AR Agonists Procaterol and 4, Leading to Novel Dopaminergics

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in mice.9 Preferential activation of one signaling pathway over the other does not only occur in response to genetically modified receptors but also in a ligand-dependent fashion. This ligand-directed signaling is known as “functional selectivity”, “ligand bias”, or “biased agonism” and has gained considerable interest over the past decades.10,11 Very recently, it was demonstrated in various animal models of PD, that dopamine receptor-mediated activation of βarrestin-2 is required for the therapeutic effects of L-DOPA. In contrast, genetic deletion of the signal transducer molecule markedly enhanced dyskinesia-like effects of both acute and chronic L-DOPA treatment and significantly reduced forward locomotion.12 Similar results were obtained when GRK6, a kinase upstream in the β-arrestin-2 signaling cascade, was deleted.13 Noteworthy, overexpression of β-arrestin-2 in PD animal models enhanced the therapeutic effects of L-DOPA while alleviating dyskinesia.12 Specifically targeting the D2Rmediated activation of β-arrestin-2 might therefore be a valuable approach for the design of novel antiparkinsonian drugs. Here we report on structural modifications of aripiprazole, a weak D2R partial agonist, leading to novel βarrestin-biased D2R agonists. Previously, a number of βarrestin-2 biased partial agonists for D2Rs including prototypic ligands 1 (UNC0006),14 2 (UNC9975).14 and 3 (UNC9994)14 have been described as putative novel antipsychotics (Chart 1). However, these ligands displayed relatively weak intrinsic activities and none of them exhibited dopamine-like properties for the recruitment of β-arrestin-2. Unlike these partial agonists, our results show hydroxy-substituted heteroarylpiperazines capable to fully activate the D2R-promoted recruitment of βarrestin-2 while displaying considerably lower tendency to stimulate G protein mediated signaling.

RESULTS AND DISCUSSION Ligand Design. When aiming to develop functionally selective agonists, a ligand-based strategy suggests starting from a balanced agonist and trying to replace or remove molecule parts associated with receptor activation. If such a modification disrupts one or the other signaling pathway more effectively, ligand bias will be observed. A complementary design embarks on an antagonist scaffold and depends on the inclusion of agonist-type structural moieties. Thus, biased agonists may be accessed by designing hybrid structures containing agonist and antagonist moieties. Starting from the typical 1,4-disubstituted aromatic piperazine (1,4-DAP) aripiprazole, a typical weak D2R partial agonist, we envisioned incorporation of typical catechollike agonist substructures in order to generate putatively biased D2R agonists. The head groups of typical 1,4-DAPs are not appropriately substituted to form hydrogen bonds with the side chains of Ser5.42, Ser5.43, and Ser5.46 within transmembrane helix 5, which are crucial for the activation of aminergic GPCRs by catecholamines and heterocyclic bioisosteres thereof.15−18 Inspired by such H-bond forming head groups,19 we conceived a formal structural hybridization of the 1,4-DAP substructure of aripiprazole and the heterocyclic neurotransmitter surrogates present in the high affinity β2-AR agonists indacaterol,20 procaterol,21,22 olodaterol,23 and BI-167107 (4),17,24 leading to a novel chemotype of dopaminergics (Chart 2). Both, the 8hydroxyquinolin-2(1H)-one and the 5-hydroxy-2H-benzo[b][1,4]oxazin-3(4H)-one substructures have been shown to mimic the phenolic hydroxyls of the endogenous β2-AR agonists norepinephrine.17,25 Besides these two heterocycles, we included the respective oxygen-analogue 8-hydroxycoumarine, an 8-hydroxy-substituted quinoline and a formally reduced 1,4-benzodioxane. Although benzodioxane substructures have been employed previously within heterocyclic 1,4-DAPs,26 4694



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Scheme 1. Synthesis of Heterocyclic Aromatic Piperazine and Homopiperazine Head Groupsa

a

Reagents and conditions: (a) DBU, benzyl bromide, isopropyl alcohol, rt, 1.5 h (70−88%); (b) Br2, AcOH, rt, 4 h (53−84%); (c) K2CO3, benzyl bromide, DMF, 50 °C, 24 h or rt, 2 h (57−91%); (d) Pd2(dba)3 or Pd(OAc)2, rac-BINAP, NaOtBu, 1-boc-piperazine or 1-boc-homopiperazine, toluene, 85 °C, 115 °C or reflux, 24−48 h (63−73%); (e) TFA, CH2Cl2, rt, 2 h (51−97%); (f) Br2, THF, 0 °C, 2 h, then rt, 16 h (47%); (g) Pd2(dba)3 or Pd(OAc)2, rac-BINAP, Cs2CO3, 1-boc-piperazine or 1-boc-homopiperazine, toluene, 85 °C or reflux, 24−72 h (38−66%); (h) Br2, CH2Cl2, rt, 5 h (55−75%).

these ligands were lacking the additional hydroxyl functionality. In addition to the variation of the aromatic head group, we

intended to replace the typical 1,4-piperazine by a homologous 1,4-diazepane (1,4-homopiperazine) because this modification 4695



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Scheme 2. Synthesis of Full-Length Dopaminergicsa

a Reagents and conditions: (a) 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)-one, NaI, acetonitrile, reflux 30 min, then addition of triethylamine in acetonitrile, 80 °C, 16 h (45−74%); (b) 5-(4-bromobutoxy)-pyrazolo[1,5-a]pyridine, NaI, acetonitrile, reflux 30 min, then addition of triethylamine in acetonitrile, 80 °C, 16 h (65−77%); (c) 10% Pd/C in acetic acid/methanol, rt, 4 h (63−79%) or TFA in toluene, reflux, 8 h (57−79%) or MsOH in toluene, reflux, 3 h (48−59%).

Therefore, a second O-benzylation was performed, yielding the 2,8-bis(benzyloxy)-5-bromoquinoline 5d. The synthesis of 8-hydroxycoumarine-based ligands was established in analogy to described procedures,31,32 starting from 2,3-dihydroxybenzaldehyde, which was subjected to a Wittig reaction and intramolecular cyclization with carbethoxycarbonylmethylene triphenylphosphorane in diethylaniline. The phenol 6a was subsequently converted into the benzyl ether 6b, and reaction with bromine in THF yielded the central building block 8(benzyloxy)-5-bromo-2H-chromen-2-one (6c). The 5-hydroxy2H-1,4-benzoxazin-3(4H)-one 7a was prepared by following a previously described three step procedure. First, 2-nitroresorcinol was converted to 2-aminoresorcinol by catalytic hydrogenation. The primary aromatic amine was reacted with chloroacetyl chloride before the final cyclization under basic conditions afforded 7a.19 Subsequently, the phenol group was reacted with benzyl bromide using DBU as a base and the aromatic core was brominated in the para-position of the benzyl-protected hydroxyl, giving access to the intermediates 7b and 7c.19 The lactam nitrogen required protection by addition of a second benzyl group, which was introduced by reaction with benzyl bromide, using elevated temperatures and potassium carbonate as a base to afford the intermediate 7d. Synthesis of the 8-hydroxybenzo-1,4-dioxane head group started from pyrogallol, which was converted to 8-hydroxybenzo-1,4-dioxane (8a) by reaction with 1,2-dibromoethane following a previously described protocol.33 Subsequently, the phenolic hydroxyl group was benzyl-protected (8b) and a

has been previously reported to foster D2R activation in a putatively β-arrestin-biased manner.14,27 As lipophilic appendages, we either employed the 3,4-dihydroquinolin-2(1H)-one substructure of aripiprazole or used a pyrazolo[1,5-a]pyridine, a moiety that has been previously shown to convey high dopamine receptor affinity.28,29 In both cases, we connected the lipophilic moiety to the piperazine in analogy to the lead structure aripiprazole by using a flexible butoxy-spacer. Synthesis. Synthesis of heteroarylpiperazines comprising a 3,4-dihydroquinolin-2(1H)-one or pyrazolo[1,5-a]pyridine moiety and a butoxy spacer was established based on a convergent synthesis strategy. In general, heteroarylpiperazines and their homopiperazine analogues were synthesized starting from the unsaturated heterocycles, which were benzylprotected and brominated to allow introduction of N-bocprotected 1,4-piperazine or 1,4-diazepane building blocks by Buchwald−Hartwig amination reactions (Scheme 1). These head groups were then reacted with the 4-bromobutoxysubstituted 3,4-dihydroquinolin-2(1H)-one or pyrazolo[1,5a]pyridine moieties to give the target compounds (Scheme 2). In detail, the synthesis of the 8-(benzyloxy)-5-bromoquinolin-2(1H)-one building block 5c25,30 was performed starting from commercially available 8-hydroxyquinolin-2-(1H)-one which was reacted with benzyl bromide using DBU as a base followed by bromination of the aromatic core in the paraposition of the benzyl-protected hydroxy group, yielding the intermediates 5b and 5c, respectively. Unfortunately, reaction of 5c and boc-protected piperazine was not successful. 4696



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Scheme 3. Synthesis of Heteroarylpiperazines with Alternate Appendagesa

a

Reagents and conditions: (a) 4-(3-(1-butyl-1H-1,2,3-triazol-4-yl)propoxy)-N-(3-oxopropyl)-3-methoxybenzamide or 4-(3-(1-butyl-1H-1,2,3-triazol4-yl)propoxy)-N-(4-oxobutyl)-3-methoxybenzamide, NaBH(OAc)3, dichloroethane, rt, 6 h (80−89%); (b) MsOH in toluene, reflux, 3 h (43−74%).

bromine residue was introduced into the para-position of the benzyl-protected hydroxyl, yielding intermediate 8c. Benzyl protection34 followed by bromination of commercially available 8-hydroxyquinoline afforded 9c,35 which served as precursor for 8-hydroxyquinoline-derived ligands. The five different bromosubstituted heterocycles were subsequently reacted with bocprotected 1,4-piperazine in Buchwald−Hartwig cross-coupling reactions, using conditions described for the reaction of 9c with piperidine.35 All reactions were carried out in toluene using either Cs2CO3 or NaOtBu as a base. As palladium species, Pd(OAc)2 or Pd2(dba)3 together with racemic BINAP were used. Using the reaction sequence elaborated for the preparation of the arylpiperazines 5e, 6d, 7e, 8d, and 9d, reaction of the bromo-substituted precursors 5d, 6c, and 7d with boc-protected 1,4-diazepane yielded the homopiperazine analogues 5f, 6e, and 7f. Finally, cleavage of the tertbutyloxycarbonyl groups with TFA in dichloromethane resulted in formation of five 1,4-heteroarylpiperazines (5g, 6f, 7g, 8e, 9e) and three 1,4-heteroarylhomopiperazines (5h, 6g, 7h). For the synthesis of the final compounds, the secondary amines of the heteroarylpiperazines (5g, 6f, 7g, 8e, 9e) and -homopiperazines (5h, 6g, 7h) were subjected to nucleophilic displacement reactions with 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)one 36 or 5-(4-bromobutoxy)pyrazolo[1,5-a]pyridine 28,37 (Scheme 2). In both cases, the alkyl bromides were activated by refluxing with sodium iodide.28,36 Finally, the remaining benzyl protecting groups were cleaved by hydrogenation or under acidic conditions, affording the heteroarylpiperazines 12a−e, 16a−e, and heteroarylhomopiperazines 13a−c and 17a. In addition to the collection of ligands bearing a secondary moiety attached via a butoxy spacer, we intended to complement our investigations with a different lipophilic appendage (Scheme 3). Very recently, we have developed a vanillin-derived scaffold linked to an amidopropyl or amidobutyl spacer as a useful moiety for specific molecular tools,38,39 and functionally selective D2R ligands.30 Thus, we ligated the homopiperazine-8-hydroxyquinolin-2(1H)-one 5h as a representative head group with the carbaldehyde function of the vanillin-type appendages by reductive amination.30,38 The intermediates 18a,b were converted to the final compounds 19a,b by cleavage of the benzyl protection groups with methanesulfonic acid in toluene. Binding Affinities. To determine the impact of the structural modifications on dopamine receptor recognition, we performed radioligand displacement studies with the dopamine receptor subtypes D1R, D2LR, D2SR, D3R, D4R, and D5R. The resulting affinity and selectivity profiles were compared to those of the chemically similar reference ligands aripiprazole and 1 (Table 1). Binding affinities were obtained

by measuring the ability of the target compounds to compete with the radioligands [3H]SCH23990 or [3H]spiperone and membranes from cells expressing the respective receptor subtypes. In general, the series of target compounds showed only weak affinity for the D1R and D5R subtypes, while they bound to the D2R-like family with low nanomolar to even subnanomolar Ki values. Indeed, heteroarylpiperazines comprising a 3,4-dihydroquinolin-2(1H)-one appendage (12a−e) were highly similar to the reference agents and displayed binding affinities ranging from 0.87 to 4.8, 0.67−2.2, and 1.0−12 nM at D2LR, D2SR and D3R, respectively. Interestingly, the incorporation of a methylene unit into the six-membered cyclic diamine (piperazine vs homopiperazine) did not significantly alter the binding affinities for ligands with 8-hydroxyquinolinone (13a) and 5-hydroxybenzoxazinone (13c) head groups (Ki 0.81−2.0 nM for D2S/LR and D3R) but induced a 15- to 40-fold loss of affinity for the homopiperazine 13b (Ki 37−240 nM) comprising an 8-hydroxycoumarine head group. When the 3,4-dihydroquinolin-2(1H)-one appendage was replaced by a pyrazolo[1,5-a]pyridine moiety (16a−e), binding affinities for D2LR and D2SR were decreased. Interestingly, the extent of this effect was found to depend on the employed heteroaryl pharmacophore. While the quinolinone 16a and the benzoxazine 16c still showed high affinity toward D2LR and D2SR (Ki 0.86−8.3 nM), derivatives lacking an the endocyclic hydrogen bond donor at the heterocyclic head group showed substantially lower binding affinities for D2LR/D2SR (Ki 19− 110 nM). The homopiperazine 17a showed lower D2R binding affinity (Ki 78 and 46 nM for D2LR and D2SR, respectively). Notably, affinities for the closely related D3R subtype did not follow the same trend, as the pyrazolo[1,5-a]pyridine derivatives 16a−c and 17a displayed low nanomolar or even subnanomolar binding affinities (Ki 0.71−8.5 nM) that were highly similar to those of the reference agents aripiprazole and 1 (Ki 1.9 and 2.9 nM). At the D4R subtype, only the heteroarylpiperazines 12c and 16c sharing the benzoxazinone head group displayed low nanomolar affinity (8.3 and 4.8 nM). Additional radioligand binding studies were performed to determine the compounds’ affinities toward related serotonergic (5-HT1AR, 5-HT2AR) and adrenergic (α1-AR) receptors using the radioligands [3H]WAY100635, [3H]ketanserin, or [ 3 H]prazosin. In comparison to the reference agents aripiprazole and 1, our newly developed compounds showed substantially altered binding properties. At the 5-HT1AR, the test compounds’ affinities ranged from 12 to 1200 nM, with the highest binding affinities observed for 12d, 12e, and 16d, containing an 8-hydroxybenzo-1,4-dioxane or an 8-hydroxyquinoline head group. 5-HT2AR affinities were substantially lower than for aripiprazole or 1, with the highest affinities observed 4697



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Table 1. Binding Affinities of the Heteroarylpiperazines Towards Dopaminergic, Serotonergic, and α-Adrenergic Receptors

a Data represent mean ± SD of 2−15 independent experiments, each performed in triplicate. bRadioligand: [3H]SCH23390. cRadioligand: [3H]spiperone. dRadioligand: [3H]WAY100635. eRadioligand: [3H]ketanserin. fRadioligand: [3H]prazosin.

was evaluated in a β-arrestin-2 recruitment assay. The arrestin receptor interaction was initially discovered to attenuate or terminate G protein-mediated signaling.41 There is now broad consensus that β-arrestins also represent signal transducers on their own.42 D2R activation mediated through β-arrestin-2 leads to the G protein-independent activation of GSK3β, an enzyme which has been implicated in hyperdopaminergic pathophysiological conditions of neuropsychiatric disorders such as schizophrenia.43 In contrast, studies with animal models of PD suggest that enhanced stimulation of β-arrestin-2 signaling may have beneficial effects on the control of locomotion while reducing dyskinesia frequently occurring in PD treatment.12 We measured the recruitment of β-arrestin-2 to ligand-activated

for the 8-hydroxyquinolines 12e and 16e and the 8hydroxybenzo-1,4-dioxane 12d. Thus, the vast majority of novel target compounds displayed high selectivity for D2R over 5-HT2AR while maintaining moderate affinity for the 5-HT1AR, a receptor that has been identified as additional target in the treatment of PD.40 Compared to the typical 1,4-DAP structures, inclusion of a propylamido- or butyramido-vanillintype appendage (19a,b) led to decreased binding affinities at D2LR, D2SR, and D4R. Notably, 19a,b displayed one-digit nanomolar Ki values at D3R (1.6 and 3.2 nM), leading to an approximately 100-fold selectivity for D3R over D2R. Functional Activity at D2R. The influence of the individual structural modifications on the ligands’ ability to activate D2R 4698



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240 nM). Notably, enlargement of the cyclic diamine moiety from a six-membered (piperazine) to a seven-membered ring (homopiperazine) increased the intrinsic efficacy of the compounds. Thus, the β-arrestin-2 recruitment assay revealed an Emax value of 75% for the homopiperazine 13c comprising a 5-hydroxybenzoxazin-3-one head group. Moreover, the 8hydroxyquinolin-2(1H)-one-homopiperazines 13a and 17a behaved as full agonists (Emax 90−102%, Figure 1a,c). These findings are in accordance with earlier reports on an increase in intrinsic efficacy for the diazepane/piperazine replacement in classical 1,4-DAP scaffolds.27 However, the presence of the homopiperazine subunit does not lead to pronounced recruitment of β-arrestin-2 on its own because the reference ligand 1 only induced marginal recruitment of β-arrestin-2 under these assay conditions (Supporting Information, Figure S1). The main difference between 1, its closely related analogues 2 and 3 (Chart 1),14,27 and our newly synthesized ligands can be found in the heterocyclic moiety were these ligands lack functional groups capable of interacting with the polar serine residues in the orthosteric binding pocket of the receptor. Similar to the rank-order in efficacies observed among the piperazines 12a−e, the 8-hydroxycoumarine-homopiperazine 13b was less effective (Emax 21%, Figure 1b) than 13a or 13c. These findings underline the importance of the presence of an additional hydrogen bond donor rather than an H-bond acceptor within the aromatic heterocycle because 13b results from 13a in a formal NH to O exchange. These results are in excellent agreement with the binding pose of the high-affinity agonist 4 within the active-state structure of the β2-AR.17 Moreover, studies on the β1-AR in complex with different agonists18 and the β2-AR crystal structure in complex with a covalent agonist25 highlighted the importance of the hydrogen bond network stabilized by catecholamine surrogates for receptor activation. All homopiperazines investigated (13a−c, 17a) displayed potencies similar to the endogenous agonist dopamine (EC50 190−470 nM). In agreement with previous results pointing toward a profitable effect of the vanillin-type appendage for the recruitment of β-arrestin-2,30 incorporation of this lipophilic moiety in combination with the 8-hydroxyquinolin-2(1H)-onesubstituted homopiperazine head group led to pronounced partial agonist properties for the recruitment of β-arrestin-2 at D2SR. However, in accordance with the reduced D2R affinities, ligands 19a,b displayed relatively weak potencies (EC50 680 and 1600 nM). In an attempt to identify β-arrestin-biased D2R agonists, a subset of the target compounds was investigated for their intrinsic activity toward the activation of canonical signaling at D2SR. Thus, we studied ligand-promoted nucleotide exchange in membrane preparations from HEK293T cells coexpressing D2SR and a pertussis toxin insensitive variant of the Gαo1 subunit44 as a proximal measure of G protein activation.45 Although D2R is known to promiscuously couple to all members of the Gαi/o family,46 Gαo proteins have been identified as predominant mediators of D2R signaling in the central nervous system (CNS).47 Incubation of the membranes with the endogenous agonist dopamine and the antiparkinsonian drugs rotigotine, ropinirole, and pramipexole led to a similar degree of nucleotide exchange compared to the reference agonist quinpirole (Table 3). While quinpirole, dopamine, pramipexole, and ropinirole showed relatively similar potencies (EC50 100−330 nM), rotigotine was effective at 30−100-fold lower concentration (EC50 3.2 nM). These results are in good agreement with the recruitment of β-

D2SR by employing the DiscoverX PathHunter technology in HEK293 cells as described previously28 and compared the results of our target compounds to those of known D2R agonists and the 1,4-DAPs aripiprazole and 1 (Table 2). When Table 2. Ligand-Induced Recruitment of β-Arrestin-2 at D2SR β-arrrestin-2 recruitment at D2SRa compd

EC50 ± SEM [nM]

Emax ± SEM [%]b

quinpirole dopamine rotigotine ropinirole pramipexole aripiprazole 1 12a 12b 12c 12d 12e 13a 13b 13c 16a 16b 16c 16d 16e 17a 19a 19b

52 ± 6 390 ± 20 4.1 ± 1.0 89 ± 5 27 ± 4 1100 ± 600 ndc 93 ± 44 ndc 92 ± 36 ndc ndc 190 ± 40 220 ± 30 470 ± 80 240 ± 60 ndc 62 ± 9 ndc ndc 370 ± 100 680 ± 70 1600 ± 600

100 ± 1 96 ± 2 101 ± 4 83 ± 4 90 ± 6 13 ± 3 ≤10 31 ± 4 ≤10 44 ± 4 ≤10 ≤10 102 ± 4 21 ± 2 75 ± 2 21 ± 1 ≤10 43 ± 3 ≤10 ≤10 90 ± 3 90 ± 2 76 ± 5

Data represent mean ± SEM of 4−10 independent experiments, each performed in duplicate. bEmax relative to the effect of the reference agonist quinpirole. cnd, not determined

a

the reference agonist quinpirole, the endogenous agonist dopamine and the antiparkinsonian drugs rotigotine, pramipexole, and ropinirole were examined for their ability to induce βarrestin-2 recruitment, sigmoid dose response curves were observed in all cases (Supporting Information, Figure S1). The ligands displayed similar efficacies ranging from full agonist behavior to very strong partial agonism observed for ropinirole (Emax 83%). The highest potency was observed for rotigotine (EC50 4.1 nM), while about 100-fold higher concentrations were necessary to activate D2SR with the endogenous agonist dopamine (EC50 390 nM). In comparison to these relatively similar effects of the reference ligands, the novel heteroarylpiperazines and their homopiperazine analogues sampled a wide range of efficacies (Supporting Information, Figure S2). Indeed, the maximum efficacy was found to depend on two main factors: the nature of the orthosteric heteroaryl moiety and the ring size of the central diamine. More specifically, piperazines 12b,d,e and 16b,d,e comprising an 8-hydroxybenzo-1,4-dioxane, an 8-hydroxyquinoline, or an 8-hydroxycoumarine head group were devoid of agonistic properties in the βarrestin-2 recruitment assay, similar to the results obtained for the antipsychotic drug aripiprazole. In contrast, ligands 12a,c and 16a,c sharing an 5-hydroxybenzoxazin-3-one or 8hydroxyquinolin-2(1H)-one substructure showed partial agonist activities with Emax values between 21% and 44% and potencies comparable to quinpirole and dopamine (EC50 62− 4699



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Figure 1. Dose−response curves at D2SR and metabolic stability of target compounds 13a,b and 17a. (a−c) Dose−response curves for D2SRmediated activation of Gαo1 (gray) and the recruitment of β-arrestin-2 in absence (blue) or presence (orange) of GRK2. (d) Bias factors for D2SR activation were calculated using the operational model of agonism. Positive values for ΔΔlog(τ/KA) indicate a preference for β-arrestin-2 recruitment. (e) Metabolic stability of 13a and 17a assessed in rat liver microsomes: distribution between metabolized substrate, substrate nonspecifically bound to matrix, and the remaining fraction after 60 min of incubation. (f) Inhibition of hERG tail currents by 13a was measured to estimate susceptibility of cardiotoxicity. Data represent mean ± SEM of at least three independent experiments.

Table 3. Ligand-Induced Nucleotide Exchange Determined with Membranes from HEK293T Cells Coexpressing D2SR and Gαo1 and β-Arrestin-2 Recruitment Promoted by D2SR in the Presence of GRK2 β-arrrestin-2 recruitment (D2SR + GRK2)a

[35S]GTPγS (D2SR + Gαo1)a compd

EC50 ± SEM [nM]

quinpirole dopamine rotigotine ropinirole pramipexole aripiprazole 1 13a 13b 13c 17a

270 ± 30 200 ± 20 3.2 ± 0.5 330 ± 60 100 ± 30 130 ± 60 >5 μMc 67 ± 20 >5 μMc 78 ± 25 90 ± 16

Emax ± SEM [%]b 100 93 91 92 102 14 17 55 36 26 56

± ± ± ± ± ± ± ± ± ± ±

EC50 ± SEM [nM]

1 2 2 2 3 2 7c 4 6c 2 4

16 160 4.4 30 9.6 740 270 200 130 630 250

± ± ± ± ± ± ± ± ± ± ±

1 20 1.1 6 1.4 180 80 20 20 110 40

Emax ± SEM [%]b 100 97 106 104 100 43 19 110 51 94 103

± ± ± ± ± ± ± ± ± ± ±

1 2 4 3 3 5 3 6 5 5 2

bias factord (D2SR) ΔΔlog(t/KA) 1.00 0.00 0.15 0.99 0.79 −0.14 2.42 −0.48 1.91 −0.58 −0.19

± ± ± ± ± ± ± ± ± ± ±

0.07e 0.08 0.10 0.08e 0.09 0.35 0.48e 0.10 0.19e 0.20 0.10

a Data represent mean ± SEM of 5−15 independent experiments, each performed in triplicate. bEmax relative to the effect of the reference agonist quinpirole. cNo complete dose response curves obtained, Emax determined at 30 μM. dLigand bias quantified by the operational model, values 0 preferential recruitment of β-arrestin-2. eSignificant bias determined by one-way ANOVA, followed by Dunnet’s post hoc test, p < 0.05.

arrestin-2, when rotigotine was also found to be the most potent agonist (EC50 4.1 nM). Relative to these high intrinsic efficacies, aripiprazole and 1 were relatively weak partial agonists (Emax 14−17%). In particular, very high concentrations of 1 were necessary to elicit guanine nucleotide exchange at D2SR. These findings are in good agreement with literature reports indicating a low tendency of 1 to inhibit cAMP formation.14 Compared to 2,3-dichlorophenyl-substituted piperazines or homopiperazines, hydroxylated heteroarylhomopiperazines 13a−c and 17a showed substantially higher intrinsic efficacies for the induction of canonical D2SR signaling (Emax 26−55%). Potencies of 13a, 13c, and 17a were found to be slightly higher than those of dopamine or quinpirole (EC50

67−90 nM), while 13b only elicited significant G protein activation at concentrations above 1 μM (Figure 1b). The target compounds 13a,c and 17a were about 50% less efficacious in promoting nucleotide exchange compared to the recruitment of β-arrestin-2 (Emax 26−55% and 75−102%, respectively). Although relative efficacies suggest a preferential engagement β-arrestins over G proteins, a quantitative bias analysis employing the operational model of agonism did not reveal significantly biased agonists among these target compounds, most likely due to the increased potencies for G protein activation. In contrast, the lactone 13b, renouncing an endocyclic hydrogen bond donor at the heterocyclic head group, was identified as a β-arrestin-biased ligand when the 4700



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Besides adequate physicochemical properties, sufficient metabolic stability is a general requirement in drug development. Thus, to determine the metabolic stability of the novel heteroarylpiperazines, we explored the representative ligands 13a and 17a for their propensity to undergo phase I transformations using rat liver microsomes in comparison to the antiparkinsonian drug rotigotine. In agreement with previous findings,53 rotigotine was extensively metabolized within 15 min of incubation, and a number of known metabolites were identified using mass spectrometry (Supporting Information, Figures S4, S5). The half-life and intrinsic clearance for rotigotine were determined to be 5.8 min and 240 μL/min/mg protein, respectively (Supporting Information, Figure S4).54,55 In contrast, only minor metabolization of the pyrazolo[1,5-a]pyridine 17a was observed (T1/2 51 min, CLint 27 μL/min/mg protein), while the 3,4-dihydroquinolin-2(1H)one 13a was hardly metabolized at all (T1/2 130 min, CLint 11 μL/min/mg protein). On the basis of the intrinsic clearance, 17a can be regarded as intermediately metabolized, while 13a falls into the low clearance category.54,55 Both ligands revealed binding to matrix protein (∼27%) in control experiments when the enzymatic transformation was prevented by the absence of the cofactor NADPH (Figure 1e, Supporting Information, Figures S4, S5). QTc-interval prolongation leading to the very rare but potentially lethal Torsades de Pointes (TdP) arrhythmia is a serious complication associated with pharmacotherapy.56,57 Although not the only cause, inhibition of the delayed rectifier potassium IKr current mediated by hERG (human-ether-à-go-go related gene) channels is predictive for QTc prolongation and the liability of a drug-like molecule to cause TdP.58 Druginduced arrhythmia has been observed for a number of dopaminergic drugs, including the phenylpiperidine haloperidol and the aromatic piperazine ziprasidone.59 To assess the susceptibility to induce arrhythmia, and hence estimate cardiotoxicity, the influence of a representative hydroxysubstituted heterocyclic phenylpiperazine (13a) on the inhibition of hERG tail currents was examined in vitro. Automated patch-clamp showed that no effect on hERG tail currents was observed at concentrations up to 1 μM (Figure 1f). At higher concentration, a slight inhibition was observed (10 μM, 71.7% current remaining), which was dose-dependently increased (50 μM, 21.5% remaining). However, the inhibitory potency (IC50) of 13a was determined to be 19.7 μM, a concentration that is 100-fold higher than the EC50 required for β-arrestin-2 recruitment at D2SR and even 24000fold higher than the binding affinity of 13a at D2SR (Ki 0.81 nM).

endogenous agonist dopamine was used as a reference (Supporting Information, Figure S3a). Bias was also detected for the very weak partial agonist 1. Because it is known that recruitment of β-arrestins is greatly facilitated upon adequate receptor phosphorylation,48 we performed additional β-arrestin-2 recruitment assays in the presence of GRK2, a kinase that has been reported to be relevant for the interaction of β-arrestin-2 and D2R.26,49,50 Dose response curves of the endogenous agonist dopamine and the reference agonists quinpirole, rotigotine, ropinirole, and pramipexole revealed highly similar maximum efficacies (Emax 97−104%) under these conditions. While the potency of rotigotine was unaffected by the presence of GRK2 (EC50 4.4 nM), a 2−3-fold increase was observed for the four other agonists including the endogenous neurotransmitter dopamine (EC50 9.6−160 nM). In good agreement with earlier reports, an increase in efficacy was observed for the weak partial agonists aripiprazole and 1 (Emax 43 and 19%, respectively). Among our newly developed hydroxylated heterocyclic homopiperazines, coexpression of GRK2 substantially increased the efficacy of 13b (Emax 51%, Figure 1b) and 13c (Emax 94%), comprising an 8-hydroxycoumarine or a 5-hydroxybenzoxazin-3-one head group, respectively. For the 8-hydroxyquinolin-2(1H)-ones 13a and 17a only minor changes in efficacy or potency were observed (Figure 1a,c), probably indicating a minor contribution of GRK2 to the signaling panorama of these two ligands. A quantitative comparison of the signaling profiles confirmed the preference for β-arrestin-2 recruitment for the 8-hydroxycoumarine 13b and the reference agent 1 (ΔΔlog(τ/KA) 1.91 and 2.42, Figure 1d). No such bias was observed for the ligands 13a,c and 17a, although the signaling profiles of these two ligands are substantially different from those of typical antiparkinsonian drugs (e.g., rotigotine) or the endogenous agonist dopamine (Supporting Information, Figures S1−3). These results are most likely caused by the detrimental changes of potency and efficacy observed for 13a,c and 17a between the different test systems and the relative importance attributed to these influencing factors in the operational model of agonism. To relate these distinct signaling profiles to pharmacological outcomes, and thus to estimate the therapeutic value of such compounds future studies, potentially also involving β-arrestin knockout animals, will be required. Metabolism and Inhibition of hERG Tail Currents. To assess the drug-like properties of our target compounds, we calculated octanol/water partition coefficients as described previously (Supporting Information, Table S1).28 Compared to the typical 1,4-DAP aripiprazole, hydroxy-substituted heteroarylpiperazines and their homopiperazine analogues were predicted to be less lipophilic. With the exception of the most hydrophilic compounds 12c, 13c, and 16c sharing the 5hydroxybenzoxazinone head group, the ligands’ lipophilicity was predicted to be similar to the range covered by the approved D2R agonists ropinirole, pramipexole, and rotigotine. Furthermore, with the exception of 19a,b, none of the compounds hit constraints predicting poor solubility or permeability as indicated by Lipinski’s rule of five.51 Metal chelating properties for various divalent cations are well-known characteristics of 8-hydroxyquinoline substructures and have been associated with undesired side effects.52 Although chelating properties are likely for some of the presented ligands, no toxic effects were observed under the conditions of our functional experiments.

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CONCLUSIONS Applied in combination with a peripheral decarboxylase inhibitor, L-DOPA remains the “gold standard” in antiparkinsonian drug therapy. However, its use is limited by wearing off and the occurrence of L-DOPA induced dyskinesia (LID) over the course of time.3 Very recently, it has been demonstrated that dopamine receptor-mediated activation of β-arrestin-2 is required for the beneficial locomotor effects of L-DOPA and not involved in the pathogenesis of LIDs. Selective targeting of this signal transducer, e.g., by biased ligands, may therefore represent a valuable novel concept in the treatment of PD.12 Aiming to develop novel dopaminergics that activate β-arrestin2 in a functionally selective manner via the interaction with D2R, we performed a formal structural hybridization of typical 4701



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for 6 min, to 10% in 3 min, then 10% for 3 min, flow rate 5 mL/min, λ = 210 or 254 nm. Preparative HPLC was performed on an Agilent 1100 Preparative series, using a Macherey-Nagel Varioprep VP 250/32 Nucleodur C18 HTec (32 mm × 250 mm, 5 μm, flow rate 32 mL/ min), a Zorbax Eclipse XDB-C8 PrepHT (21.5 mm × 150 mm, 5 μm, flow rate 10 mL/min) or a Macherey-Nagel Varioprep VP 250/10 Nucleodur C18 HTec (10 mm × 250 mm, 5 μm, flow rate 3 mL/min) column with the solvent systems indicated. 2,8-Bis(benzyloxy)-5-bromoquinoline (5d). To a solution of 5c (863 mg, 2.6 mmol) and K2CO3 (1.08 g, 2.6 mmol) in DMF (26 mL) was added dropwise benzyl bromide (0.5 mL, 3.9 mmol). The mixture was stirred at room temperature for 2 h. The solvent was evaporated, and the residue was diluted with water. The product was extracted with EtOAc, and the combined organic layers were washed with 0.5 M HCl, water, and brine, dried (Na2SO4), and evaporated. Purification by flash chromatography (hexanes/EtOAc 30:1) yielded 5d (990 mg, 91%) as a colorless solid. 1H NMR (CDCl3, 600 MHz) δ 5.31 (s, 2H), 5.58 (s, 2H), 6.96 (d, J = 8.4 Hz, 1H), 7.05 (d, J = 9.1 Hz, 1H), 7.28− 7.36 (m, 4H), 7.37−7.42 (m, 2H), 7.50 (d, J = 8.4 Hz, 1H), 7.52−7.58 (m, 4H), 8.33 (d, J = 9.1 Hz, 1H). 13C NMR (CDCl3, 151 MHz) δ 68.16, 71.55, 112.9, 113.0, 114.7, 125.4, 127.4, 127.5, 128.0, 128.1, 128.5, 128.7, 128.8, 137.2, 137.2, 138.7, 139.5, 153.2, 161.6. IR (NaCl) ν 3030, 1610, 1597, 1502, 1404, 1272, 1253, 1095, 1006, 914, 826, 732, 695 cm−1. HPLC: (system 1) tR = 23.1 min, purity >99%; (system 2) tR = 22.6 min, purity >99%. ESI-MS m/z 420.0 tert-Butyl 4-[2,8-Bis(benzyloxy)quinolin-5-yl]piperazine-1-carboxylate (5e). To a solution of 1-boc-piperazine (1.3 g, 7.2 mmol) in toluene (51 mL) were added Pd(OAc)2 (32 mg, 0.14 mmol) and rac-BINAP (286 mg, 0.43 mmol) under N2 atmosphere. After stirring for 15 min at room temperature, 5d (1.5 g, 3.6 mmol) and NaOtBu (0.5 g, 5.0 mmol) were added and the resulting mixture was stirred under reflux for 24 h. After cooling down to room temperature, EtOAc was added and the organic layer was washed with 0.5 M HCl, water, and brine, dried (Na2SO4), and evaporated. Purification by flash chromatography (hexanes/EtOAC 20:1 to 2:1) yielded 5e (1.2 g, 63%) as a colorless solid. 1H NMR (CDCl3, 600 MHz) δ 1.50 (s, 9H), 2.79−3.08 (m, 4H), 3.37−3.77 (m, 4H), 5.30 (s, 2H), 5.58 (s, 2H), 6.88 (d, J = 8.3 Hz, 1H), 6.98 (d, J = 9.0 Hz, 1H), 7.05 (d, J = 8.3 Hz, 1H), 7.28−7.33 (m, 2H), 7.33−7.40 (m, 4H), 7.47−7.67 (m, 4H), 8.39 (d, J = 9.0 Hz, 1H). 13C NMR (CDCl3, 91 MHz) δ 28.62, 53.47, 67.85, 71.98, 79.99, 112.8, 113.0, 113.5, 122.2, 127.4, 127.8, 128.0, 128.5, 128.6, 128.7, 135.2, 137.6, 137.9, 139.7, 143.6, 150.3, 155.0, 161.3. IR (NaCl) ν 2924, 2854, 1695, 1610, 1411, 1261, 1246, 1169, 1003, 734, 696 cm−1. HPLC: (system 1) tR = 23.1 min, purity >99%; (system 2) tR = 22.5 min, purity >99%. ESI-MS m/z 526.7 [M + H+]. tert-Butyl 4-[2,8-Bis(benzyloxy)quinolin-5-yl]-1,4-diazepane-1carboxylate (5f). To a solution of N-(tert-butyl-oxycarbonyl)homopiperazine (380 mg, 1.9 mmol) in toluene (15 mL), Pd(OAc)2 (8.5 mg, 0.04 mmol) and rac-BINAP (71 mg, 0.11 mmol) were added. After stirring for 15 min, 5d (400 mg, 0.95 mmol) and NaOtBu (128 mg, 1.3 mmol) were added. The tube was sealed, and the reaction mixture was stirred at 115 °C for 1 d. After cooling down to room temperature, and the reaction mixture was diluted with EtOAc, the organic layer was washed with 0.5 M HCl, water, and brine, dried (Na2SO4), and evaporated. Purification by flash chromatography (hexanes/EtOAc 40:1 to 10:1 to 2:1) yielded 1f (350 mg, 68%) as a colorless oil. 1H NMR (CDCl3, 600 MHz) δ 1.50 (d, J = 9.8 Hz, 9H), 1.86−2.13 (m, 2H), 3.02−3.27 (m, 4H), 3.55−3.73 (m, 4H), 5.30 (s, 2H), 5.58 (s, 2H), 6.94 (dd, J = 8.2, 2.6 Hz, 1H), 6.98 (d, J = 9.0 Hz, 1H), 7.00−7.06 (m, J = 8.2, 2.0 Hz, 1H), 7.28−7.33 (m, 2H), 7.33− 7.41 (m, 4H), 7.55 (dd, J = 11.8, 7.4 Hz, 4H), 8.44 (d, J = 8.8 Hz, 1H). 13 C NMR (CDCl3, 151 MHz) δ 28.69, (29.19), 29.35, (45.69), 46.48, (47.79), 48.14, (56.57), 56.97, (57.40), 57.46, 67.80, 71.98, (79.60), 79,64, (112.7), 113.1, (115.2), 115.2, 122.8, 127.4, 127.8, 128.0, 128.5, 128.6, 128.7, 135.6, (137.6), 137.6, 137.9, 139.5, (146.0), 146.1, (150.0), 150.0, (155.7), 155.8, 161.3 (signals in brackets represent resonances caused by rotamers). IR (NaCl) ν 3432, 2974, 1645, 1481, 1453, 1408, 1366, 1312, 1261, 1154, 736, 699 cm−1. HPLC: (system 1) tR = 23.0 min, purity >98%; (system 2) tR = 22.7 min, purity >99%. ESI-MS m/z 540.2 [M + H+].

1,4-DAPs and heterocyclic catecholamine surrogates leading to hydroxy-substituted heteroaryl-1,4-disubstituted piperazines and homopiperazines. In contrast to previously described βarrestin-2 biased ligands for D2R,14,27 these compounds are designed to mimic the interactions of the catechol substructure of the endogenous ligand dopamine with crucial serine residues in the orthosteric binding pocket. We found that two major factors determined the intrinsic efficacy of the ligands with this novel scaffold: the presence of an additional endocyclic H-bond donor rather than an H-bond acceptor and the ring-size of the cyclic diamine. Notably, ligands 13a,c and 17a comprising an 8hydroxyquinolin-2(1H)-one or a 5-hydroxy-2H-benzo[b][1,4]oxazin-3(4H)-one heterocycle and a homopiperazine substructure showed full agonist properties for the recruitment of β-arrestin-2 while they were substantially less efficacious for the induction of D2SR-promoted guanine nucleotide exchange as a measure of canonical Gαo protein activation. Although the 8-hydroxycoumarine-substituted homopiperazine 13b induced submaximal activation of both signaling pathways, this compound was identified as highly β-arrestin-biased using the operational model of agonism. A further evaluation of representative homopiperazines 13a and 17a comprising a 3,4-dihydroquinolin-2(1H)-one or pyrazolo[1,5-a]pyridine appendage revealed a low tendency to undergo metabolic transformation reactions in rat liver microsomes and identified a minor liability to inhibit hERG channels, an important offtarget in the course of drug development. The combination of typical agonist-type elements from known β2-AR agonists such as procaterol21,22 and 417,24 with 1,4-DAP substructures known for their weak partial agonist characteristics60 proved successful for the design of novel β-arrestin-biased D2R agonists with unprecedented intrinsic efficacy. This novel scaffold might serve as a starting point for the development of functionally selective PD therapeutics with a lower propensity to induce LID. Nevertheless, the contribution of other dopamine receptor subtypes, especially the D1R, to the efficacy and side effects associated with L-DOPA therapy should also be addressed in drug discovery.6,12 Medicinal chemistry specifically targeting D1R-promoted β-arrestin-2 recruitment may lead to further promising innovations.

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EXPERIMENTAL SECTION

Chemistry. Reagents and dry solvents were of commercial quality and used as purchased. If not stated otherwise, reactions were carried out under nitrogen atmosphere. MS was run on a BRUKER ESQUIRE 2000 or on a BRUKER amaZon SL mass spectrometer using ESI ionization. HRMS was run on a AB Sciex Triple TOF660 SCiex, source type ESI, or at the Chair of Organic Chemistry, Friedrich Alexander University Erlangen−Nuernberg on a Bruker Daltonik micrOTOF II focus or Bruker Daltonik maXis 4G, source type ESI or APPI. NMR spectra were recorded on a Bruker Avance 400, Bruker Avance 360, or a Bruker Avance 600 spectrometer at 300 K in the solvents indicated. Chemical shifts are given in ppm (δ) relative to TMS. IR spectra were obtained on a Jasco FT/IR 4100 spectrometer using a substance film on a NaCl crystal. Purification by column chromatography was performed with silica gel 60. TLC analyses were performed using Merck 60 F254 aluminum plates in combination with UV detection (254 nm) or ninhydrin staining. Analytical HPLC was conducted on an Agilent 1200 HPLC system employing a DAD detector and a Zorbax Eclipse XDB-C8 (4.6 mm × 150 mm, 5 μm) column with the following binary solvent systems: System 1, eluent, methanol/0.1% aq trifluoroacetic acid, 10% methanol for 3 min, to 100% in 15 min, 100% for 6 min, to 10% in 3 min, then 10% for 3 min, flow rate 5 mL/min, λ = 210 or 254 nm; System 2, CH3CN/0.1% aq trifluoroacetic acid, 10% CH3CN for 3 min, to 100% in 15 min, 100% 4702



Article

4-[2,8-Bis(benzyloxy)quinolin-5-yl]piperazine (5g). To a solution of 5e (550 mg, 0.95 mmol) in CH2Cl2 (13 mL) was added TFA (1.3 mL). The mixture was stirred at room temperature for 2 h. The reaction was stopped by adjusting the pH to 9 by addition of 1 M NaOH. The product was extracted with CH2Cl2, and the combined organic phases were dried over Na2SO4 and evaporated. Purification by flash chromatography (CH2Cl2/MeOH 15:1) yielded 5g (220 mg, 51%) as a yellow solid. 1H NMR (CDCl3, 360 MHz) δ 3.11 (m, 4H), 3.26 (m, 4H), 3.45 (s, 1H), 5.31 (s, 2H), 5.58 (s, 2H), 6.94 (d, J = 8.3 Hz, 1H), 6.99 (d, J = 9.0 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 7.43−7.27 (m, 6H), 7.55 (t, J = 7.3 Hz, 4H), 8.36 (d, J = 9.1 Hz, 1H). 13C NMR (CDCl3, 91 MHz) δ 45.54, 52.64, 67.89, 71.89, 112.8, 113.0, 114.0, 122.1, 127.4, 127.9, 128.0, 128.5, 128.6, 128.7, 135.0, 137.5, 137.8, 139.7, 143.0, 150.6, 161.3. IR (NaCl) ν 3439, 3031, 2946, 1610, 1411, 1264, 1005, 742, 693 cm−1. HPLC: (system 1) tR = 19.9 min, purity 99%; (system 2) tR = 16.5 min, purity 93%. ESI-MS m/z 426.1 [M + H+]. 4-[2,8-Bis(benzyloxy)quinolin-5-yl]-1,4-diazepane (5h). Compound 5h was prepared as described for 5g, using a mixture of 5f (200 mg, 0.37 mmol) and TFA (0.5 mL) in CH2Cl2 (5.0 mL). Purification by flash chromatography (CH2Cl2/MeOH 15:1) yielded 5h (129 mg, 79%) as a yellow solid. 1H NMR (CDCl3, 600 MHz) δ 1.95−2.12 (m, 2H), 3.09−3.40 (m, 8H), 5.30 (s, 2H), 5.58 (s, 2H), 6.94−7.01 (m, 2H), 7.04 (d, J = 8.4 Hz, 1H), 7.27−7.32 (m, 2H), 7.33−7.40 (m, 4H), 7.49−7.58 (m, 4H), 8.46 (d, J = 9.0 Hz, 1H). 13C NMR (CDCl3, 91 MHz) δ 29.97, 46.92, 49.40, 56.66, 57.79, 67.82, 72.02, 112.8, 113.1, 115.4, 122.8, 127.4, 127.8, 127.9, 128.5, 128.6, 128.7, 135.6, 137.6, 137.9, 139.6, 145.9, 150.1, 161.3. IR (NaCl) ν 3432, 3034, 2937, 2848, 1650, 1559, 1506, 1411, 1260, 1093, 733, 697 cm−1. HPLC: (system 1) tR = 20.0 min, purity 87%; (system 2) tR = 16.8 min, purity 91%. ESI-MS m/z 440.2 [M + H+]. tert-Butyl 4-[8-(Benzyloxy)-2-oxo-2H-chromen-5-yl]piperazine-1carboxylate (6d). Pd2(dba)3 (24 mg, 0.03 mmol) and rac-BINAP (49 mg, 0.08 mmol) were dissolved in toluene (9 mL) and stirred for 5 min under nitrogen atmosphere. Then Cs2CO3 (300 mg, 0.92 mmol), 1-boc-piperazine (307 mg, 1.6 mmol), and 6c (220 mg, 0.66 mmol) were added, and the reaction mixture was stirred in a sealed microwave reaction tube at 85 °C for 1 d. After cooling down to room temperature, the reaction was diluted and EtOAc was added. The combined organic layer was washed with 0.5 M HCl, water, and brine, dried (Na2SO4) and evaporated. Purification by flash chromatography (hexanes/EtOAc 20:1 to 5:1) yielded 6d (172 mg, 59%) as a yellow solid. 1H NMR (CDCl3, 600 MHz) δ 1.49 (s, 9H), 2.75−3.08 (m, 4H), 3.45−3.88 (m, 4H), 5.20 (s, 2H), 6.43 (d, J = 9.8 Hz, 1H), 6.80 (d, J = 8.7 Hz, 1H), 7.03 (d, J = 8.7 Hz, 1H), 7.32 (t, J = 7.3 Hz, 1H), 7.38 (t, J = 7.5 Hz, 2H), 7.47 (d, J = 7.4 Hz, 2H), 8.07 (d, J = 9.8 Hz, 1H). 13C NMR (CDCl3, 91 MHz) δ 28.58, 44.16, 53.55, 71.85, 80.20, 114.7, 115.4, 116.0, 116.9, 127.5, 128.2, 128.8, 136.6, 140.1, 143.0, 143.9, 145.5, 154.9, 160.3. IR (NaCl) ν 2975, 2917, 2857, 1727, 1690, 1487, 1454, 1424, 1267, 1250, 1170, 1127, 1069, 1011, 908, 810 cm−1. HPLC: (system 1) tR = 21.2 min, purity 94%; (system 2) tR = 19.8 min, purity 99%. ESI-MS m/z 459.2 [M + Na+]. tert-Butyl 4-[8-(Benzyloxy)-2-oxo-2H-chromen-5-yl]-1,4-diazepane-1-carboxylate (6e). Compound 6e was prepared according to the protocol of 6d, using a solution of Pd2(dba)3 (24 mg, 0.03 mmol), rac-BINAP (49 mg, 0.08 mmol), Cs2CO3 (300 mg, 0.92 mmol), N(tert-butyl-oxycarbonyl)-homopiperazine (307 mg, 1.6 mmol), and 6c (220 mg, 0.66 mmol) in toluene (9 mL). Purification by flash chromatography (hexanes/EtOAc 20:1 to 5:1) yielded 6e (172 mg, 59%) as a yellow solid. 1H NMR (CDCl3, 600 MHz) δ 1.46−1.53 (m, 9H), 1.88−2.04 (m, 2H), 3.03−3.16 (m, 4H), 3.52−3.59 (m, 2H), 3.59−3.70 (m, 2H), 5.20 (s, 2H), 6.41 (d, J = 9.7 Hz, 1H), 6.85 (dd, J = 8.7, 4.0 Hz, 1H), 7.00 (dd, J = 8.6, 3.6 Hz, 1H), 7.31−7.33 (m, 1H), 7.36−7.40 (m, 2H), 7.45−7.48 (m, 2H), 8.07−8.14 (m, 1H). 13C NMR (CDCl3, 101 MHz) δ 28.50, (28.85), 29.08, (45.46), 46.33, (47.25), 47.66, (56.34), 56.75, 57.19, 71.67, 115.6, 116.3, 116.8, 127.4, 128.0, 128.6, 129.5, 134.0, 136.5, 140.5 (signals in brackets represent resonances caused by rotamers) IR (NaCl) ν 3416, 2974, 2927, 1730, 1655, 1487, 1264, 1070 cm−1. HPLC: (system 1) tR = 21.5 min, purity

96%; (system 2) tR = 20.1 min, purity 96%. ESI-MS m/z 451.1 [M + H+]. 4-[8-(Benzyloxy)-2-oxo-2H-chromen-5-yl]piperazine (6f). Compound 6f was prepared as described for 6g, using a mixture of 6d (170 mg, 0.39 mmol) and TFA (0.3 mL) in CH2Cl2 (5.3 mL). Purification by flash chromatography (CH2Cl2/MeOH 15:1) yielded 6f (85 mg, 65%) as a yellow solid. 1H NMR (DMSO-d6, 360 MHz) δ 2.71−2.87 (m, 4H), 2.87−3.03 (m, 4H), 5.20 (s, 2H), 6.46 (d, J = 9.8 Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H), 7.30 (d, J = 8.8 Hz, 1H), 7.34−7.38 (m, 1H), 7.38−7.45 (m, 2H), 7.46−7.52 (m, 2H), 8.11 (d, J = 9.8 Hz, 1H). 13C NMR (DMSO-d6, 151 MHz) δ 45.53, 54.00, 70.45, 114.2, 114.3, 115.1, 116.3, 127.8, 128.0, 128.5, 136.7, 140.7, 141.6, 144.2, 159.5. IR (NaCl) ν 3432, 1684, 1377, 1272, 1204, 1131, 1060 cm−1. HPLC: (system 1) tR = 16.9 min, purity 99%; (system 2) tR = 14.0 min, purity 99%. ESI-MS m/z 337.2 [M + H+]. 4-[8-(Benzyloxy)-2-oxo-2H-chromen-5-yl]-1,4-diazepane (6g). Compound 6g was prepared as described for 5g, using a mixture of 6e (87 mg, 0.19 mmol) and TFA (0.25 mL) in CH2Cl2 (2.50 mL). Purification by flash chromatography (CH2Cl2/MeOH 15:1) yielded 6g (38 mg, 57%) as a yellow solid. 1H NMR (CDCl3, 400 MHz) δ 1.91−2.02 (m, 2H). 3.08−3.13 (m, 2H), 3.13−3.21 (m, 2H), 3.22− 3.28 (m, 2H), 3.29−3.41 (m, 2H), 5.19 (d, J = 8.3 Hz, 1H), 6.48 (d, J = 9.8 Hz, 1H), 7.04 (d, J = 8.8 Hz, 1H), 7.31 (d, J = 8.9 Hz, 1H), 7.34−7.38 (m, 1H), 7.39−7.45 (m, 2H), 7.46−7.52 (m, 2H), 8.24 (d, J = 9.8 Hz, 1H). 13C NMR (CDCl3, 101 MHz) δ 27.33, 45.12, 47.02, 54.48, 55.86, 70.42, 115.0, 115.2, 116.3, 116.5, 127.8, 128.0, 128.5, 136.6, 141.2, 141.7, 143.9, 145.3, 159.5. IR (NaCl) ν 3421, 2959, 2922, 2850, 1722, 1651, 1488, 1454, 1270, 1192, 1134, 1067, 806, 699 cm−1. HPLC: (system 1) tR = 17.1 min, purity 91%; (system 2) tR = 14.2 min, purity 88%. ESI-MS m/z 351.1 [M + H+]. 4-Benzyl-5-(benzyloxy)-8-bromo-2H-benzo[b][1,4]oxazin-3(4H)one (7d). To a solution of 7c (3.7 g, 11.0 mmol) and K2CO3 (4.5 g, 33.0 mmol) in DMF (110 mL) was added dropwise benzyl bromide (1.9 mL, 16.5 mmol). The mixture was stirred at room temperature for 2 h. The solvent was evaporated, and the residue was diluted with water. The product was extracted with EtOAc, and the combined organic layers were washed with 0.5 M HCl, water, and brine, dried (Na2SO4), and evaporated. Purification by flash chromatography (hexanes/EtOAc 12:1 to 1:1) yielded 7d (2.67 g, 57%) as a colorless solid. 1H NMR (DMSO-d6, 600 MHz) δ 4.65 (s, 2H), 4.95 (s, 2H), 5.37 (s, 2H), 6.50 (d, J = 9.0 Hz, 1H), 6.99 (d, J = 7.1 Hz, 2H), 7.13 (d, J = 9.0 Hz, 1H), 7.14−7.20 (m, 3H), 7.28−7.32 (m, 2H), 7.34− 7.41 (m, 3H). 13C NMR (DMSO-d6, 151 MHz) δ 46.89, 69.24, 71.63, 102.8, 109.2, 120.2, 127.0, 127.1, 127.7, 128.2, 128.4, 128.6, 128.9, 135.8, 137.0, 146.7, 148.8, 166.2. IR (NaCl) ν 1692, 1485, 1456, 1386, 1279, 1103, 1016, 739, 702 cm−1. HPLC: (system 1) tR = 21.7 min, purity 89% (system 2) tR = 20.2 min, purity 87%. ESI-MS m/z 425.9 [M + H+]. tert-Butyl 4-[4-Benzyl-5-(benzyloxy)-2H-benzo[b][1,4]oxazin3(4H)-one-8-yl]piperazin-1-carboxylate (7e). To a solution of 1boc-piperazine (385 mg, 2.1 mmol) in toluene (13 mL) were added Pd(OAc)2 (8.5 mg, 0.04 mmol) and rac-BINAP (70 mg, 0.13 mmol) under nitrogen atmosphere. After stirring for 15 min, 7d (400 mg, 0.94 mmol) and Cs2CO3 (1.1 g, 3.3 mmol) were added. The resulting mixture heated to reflux for 72 h. After cooling down to room temperature, the reaction mixture was diluted with EtOAc, and the organic layer was washed with 0.5 M HCl, water, and brine, dried (Na2SO4), and evaporated. Purification by flash chromatography (H/ EtOAC 12:1 to 2:1) yielded 7e (313 mg, 66%) as a light-brown solid. 1 H NMR (CDCl3, 360 MHz) δ 1.48 (s, 9H), 2.80−2.95 (m, 4H), 3.49−3.65 (m, 4H), 4.57 (s, 2H), 4.92 (s, 2H), 5.39 (s, 2H), 6.52 (d, 1H, J = 9.0 Hz), 6.57 (d, 1H, J = 9.0 Hz), 6.95−7.05 (m, 2H), 7.08− 7.21 (m, 3H), 7.28−7.51 (m, 5H). 13C NMR (CDCl3, 91 MHz) δ 28.60, 46.90, 51.25, 68.95, 71.61, 79.94, 107.8, 114.5, 120.2, 127.0, 127.0, 127.7, 128.3, 128.4, 128.8, 135.9, 136.4, 137.40, 142.9, 145.4, 154.9, 166.6. IR (NaCl) ν 2923, 2852, 1688, 1502, 1390, 1245, 1169, 1101, 1023, 911, 732 cm−1. HPLC: (system 1) tR = 21.8 min, purity 89% (system 2) tR = 19.9 min, purity 91%. ESI-MS m/z 530.1 [M + H+]. 4703



Article

1-[8-(Benzyloxy)-2,3-dihydrobenzo[b][1,4]dioxin-5-yl]piperazine (8e). Compound 8e was prepared according to the protocol of 5g, using a solution of 8d (450 mg, 1.05 mmol) in CH2Cl2 (12 mL) and TFA (1.2 mL). Purification by flash chromatography (CH2Cl2/MeOH 15:1) yielded 8e (214 mg, 66%) as a colorless solid. 1H NMR (CDCl3, 600 MHz) δ 2.99−2.96 (m, 4H), 3.10−3.06 (m, 4H), 4.33−4.30 (m, 2H), 4.35−4.33 (m, 2H), 5.09 (s, 2H), 6.41 (d, J = 8.9 Hz, 1H), 6.45 (d, J = 8.9 Hz, 1H), 7.29 (t, J = 7.3 Hz, 1H), 7.36 (dd, J = 10.3, 4.7 Hz, 2H), 7.43 (d, J = 7.5 Hz, 2H). 13C NMR (CDCl3, 91 MHz) δ 46.18, 52.19, 64.21, 64.36, 71.61, 105.8, 109.2, 127.6, 128.0, 128.6, 134.9, 136.2, 137.4, 137.7, 144.3. IR(NaCl) ν 3396, 2926, 2873, 1501, 1471, 1278, 1239, 1112, 996, 789 cm−1. HPLC: (system 1) tR = 16.6 min, purity 93%; (system 2) tR = 13.5 min, purity 95%. ESI-MS m/z 327.1 [M + H+]. tert-Butyl 4-[8-(Benzyloxy)quinolin-5-yl]piperazine-1-carboxylate (9d). To a solution of 1-boc-piperazine (391 mg, 2.1 mmol) in toluene (15 mL), Pd(OAc)2 (9 mg, 0.04 mmol) and rac-BINAP (0.13 mmol, 86 mg) were added. After stirring for 15 min, 9c (330 mg, 1.0 mmol) and NaOtBu (141 mg, 1.47 mmol) were added. The tube was sealed, and the reaction mixture was stirred at 115 °C for 1 d. After cooling down to room temperature, the reaction mixture was diluted with EtOAc, and the organic layer was washed with 0.5 M HCl, water, and brine, dried (Na2SO4), and evaporated. Purification of the crude product by flash chromatography (H/EtOAc 20:1 to 2:1) yielded 9d (320 mg, 73%) as a yellow solid. 1H NMR (CDCl3, 600 MHz) δ 1.50 (s, 9H), 2.72−3.18 (m, 4H), 3.31−4.22 (m, 4H), 5.42 (s, 2H), 6.94 (d, J = 8.3 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 7.30 (t, J = 7.4 Hz, 1H), 7.37 (dd, J = 10.4, 4.7 Hz, 2H), 7.46 (dd, J = 8.5, 4.2 Hz, 1H), 7.49−7.55 (m, 2H), 8.55 (dd, J = 8.5, 1.7 Hz, 1H), 8.99 (dd, J = 4.2, 1.7 Hz, 1H). 13 C NMR (CDCl3, 151 MHz) δ 28.59, 44.43, 53.37, 70.98, 80.01, 109.7, 115.8, 121.2, 125.5, 127.2, 127.9, 128.7, 132.0, 137.3, 141.3, 142.4, 149.4, 151.2, 155.0. IR (NaCl) ν 2974, 2928, 2858, 2815, 1691, 1608, 1454, 1365, 1247, 1170, 1103, 1008, 912, 734 cm−1. HPLC: (system 1) tR = 19.1 min, purity 99% (system 2) tR = 16.3 min, purity 99%. ESI-MS m/z 420.4 [M + H+]. 4-[8-(Benzyloxy)quinolin-5-yl]piperazine (9e). Compound 9e was prepared following the protocol of 5g, using a mixture of 9d (600 mg, 1.43 mmol) and TFA (1.8 mL) in CH2Cl2 (18 mL). Purification by flash chromatography (CH2Cl2/MeOH 15:1) yielded 9e (443 mg, 97%) as a light-yellow solid. 1H NMR (CDCl3, 600 MHz) δ 2.97−3.08 (m, 4H), 3.16 (t, J = 4.8 Hz, 4H), 5.42 (s, 2H), 6.94 (d, J = 8.3 Hz, 1H), 7.00 (d, J = 8.3 Hz, 1H), 7.29 (t, J = 7.4 Hz, 1H), 7.33−7.38 (m, 2H), 7.45 (dd, J = 8.5, 4.1 Hz, 1H), 7.51 (d, J = 7.2 Hz, 2H), 8.53 (dd, J = 8.5, 1.7 Hz, 1H), 8.98 (dd, J = 4.1, 1.7 Hz, 1H). 13C NMR (CDCl3, 151 MHz) δ 46.42, 54.23, 71.00, 109.7, 115.7, 121.1, 125.5, 127.2, 127.9, 128.7, 132.1, 137.4, 141.4, 142.9, 149.4, 151.1. IR (NaCl) ν 3416, 3062, 2943, 2821, 1607, 1472, 1364, 1304, 1253, 1099, 911, 816, 792, 734 cm−1. HPLC: (system 1) tR = 14.2 min, purity 99% (system 1) tR = 11.5 min, purity 99%. ESI-MS m/z 320.3 [M + H+], 342.2 [M + Na+]. 7-(4-{4-[2,8-Bis(benzyloxy)quinolin-5-yl]piperazin-1-yl}butoxy)3,4-dihydroquinolin-2(1H)-one (10a). A solution of 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)-one (214 mg, 0.72 mmol) and NaI (162 mg, 1.08 mmol) in acetonitrile (3.6 mL) was heated to for 0.5 h before a solution 5g (400 mg, 0.94 mmol) and triethylamine (150 μL, 1.08 mmol) in acetonitrile (1.8 mL) was added. The reaction mixture was stirred at 80 °C for further 16 h. After cooling down to room temperature, saturated NaHCO3 solution was added and the product was extracted with CH2Cl2. The combined organic layers were dried (Na2SO4) and evaporated. Purification by flash chromatography (CH2Cl2/MeOH 40:1 to15:1) yielded 10a (263 mg, 57%) as a colorless solid. 1H NMR (CDCl3, 600 MHz) δ 1.68−1.78 (m, 2H), 1.78−1. 90 (m, 2H), 2.49−2.56 (m, 2H), 2.61 (dd, J = 8.2, 6.8 Hz, 2H), 2.65−2.86 (m, 4H), 2.83−2.94 (m, 2H), 2.96−3.12 (m, 4H), 3.97 (t, J = 6.3 Hz, 2H), 5.30 (s, 2H), 5.58 (s, 2H), 6.33 (d, J = 2.4 Hz, 1H), 6.53 (dd, J = 8.3, 2.4 Hz, 1H), 6.91 (d, J = 8.3 Hz, 1H), 6.97 (d, J = 9.0 Hz, 1H), 7.02−7.08 (m, 2H), 7.27−7.33 (m, 2H), 7.33−7.43 (m, 4H), 7.50−7.60 (m, 4H), 8.07 (s, 1H), 8.38 (d, J = 9.0 Hz, 1H). 13C NMR (DMSO-d6, 151 MHz) δ 23.58, 24.75, 27.43, 31.25, 53.48, 53.89, 58.45, 67.80, 68.06, 71.99, 102.3, 108.8, 112.5, 113.1, 113.2,

tert-Butyl 4-[4-Benzyl-5-(benzyloxy)-3-oxo-3,4-dihydro-2Hbenzo[b][1,4]oxazin-8-yl]-1,4-diazepane-1-carboxylate (7f). Compound 7f was prepared as described for 7e, using a solution of N-(tertbutyl-oxycarbonyl)-homopiperazine (467 mg, 2.3 mmol), Pd(OAc)2 (9.5 mg, 0.04 mmol), rac-BINAP (74 mg, 0.12 mmol), 7d (450 mg, 1.1 mmol), and Cs2CO3 (1.21 g, 3.7 mmol) in toluene (15 mL), allowing a reaction time of 48 h. Purification by flash chromatography (hexanes/EtOAc 12:1 to 2:1) yielded 7f (218 mg, 38%) as a lightbrown solid. 1H NMR (CDCl3, 600 MHz, incl rotamers) δ 1.45 (d, J = 10.8 Hz, 9H), 2.01−1.81 (m, 2H), 3.32−3.02 (m, 4H), 3.67−3.36 (m, 4H), 4.53 (s, 2H), 4.90 (d, J = 3.6 Hz, 2H), 5.38 (s, 2H), 6.48 (dd, J = 8.8, 4.9 Hz, 1H), 6.59 (d, J = 9.0 Hz, 1H), 7.07−6.99 (m, 2H), 7.19− 7.10 (m, 3H), 7.32−7.29 (m, 2H), 7.39−7.32 (m, 3H). 13C NMR (CDCl3, 91 MHz) δ 21.17, 28.65, 45.92, 46.92, 48.06, 53.65, 54.17, 68.92, 71.71, 79.48, 108.0, 115.1, 120.3, 126.9, 127.1, 127.7, 128.3, 128.8, 136.6, 136.7, 137.5, 142.4, 144.4, 155.6, 166.8. IR (NaCl) ν 3026, 2946, 2830, 1686, 1503, 1454, 1385, 1267, 1023, 738, 695 cm−1. HPLC: (system 1) tR = 21.8 min, purity 74%. ESI-MS m/z 544.2 [M + H+]. 4-[4-Benzyl-5-(benzyloxy)-2H-benzo[b][1,4]oxazin-3(4H)-one-8yl]piperazine (7g). Compound 7g was prepared as described for 5g, using a mixture of 7e (255 mg, 0.48 mmol) and TFA (0.6 mL) in CH2Cl2 (6.4 mL). Purification by flash chromatography (CH2Cl2/ MeOH 15:1) yielded 7g (161 mg, 87%) as a light-yellow solid. 1H NMR (CDCl3, 600 MHz) δ 3.00−2.95 (m, 4H), 3.13−3.03 (m, 4H), 4.57 (s, 2H), 4.92 (s, 2H), 5.39 (s, 2H), 6.53 (d, J = 9.0 Hz, 1H), 6.60 (d, J = 9.0 Hz, 1H), 7.03−6.99 (m, 2H), 7.18−7.10 (m, 3H), 7.32− 7.29 (m, 2H), 7.39−7.33 (m, 3H). 13C NMR (CDCl3, 151 MHz) δ 46.08, 46.85, 52.15, 68.89, 71.54, 107.8, 114.4, 120.1, 126.9, 127.0, 127.7, 128.3, 128.3, 128.8, 136.4, 136.4, 137.4, 142.8, 145.2, 166.7. IR (NaCl) ν 3432, 2851, 1655, 1504, 1457, 1389, 1258, 1101, 1262 cm−1. HPLC: (system 1) tR = 19.0 min, purity 99% (system 2) tR = 15.2 min, purity 96%. ESI-MS m/z 430.0 [M + H+]. 4-[4-Benzyl-5-(benzyloxy)-2H-benzo[b][1,4]oxazin-3(4H)-one-8yl]-1,4-diazepane (7h). Compound 7h was prepared according to the protocol of 5g, using a mixture of 7f (156 mg, 0.29 mmol) and TFA (0.4 mL) in CH2Cl2 (4.0 mL). Purification by flash chromatography (CH2Cl2/MeOH 15:1) yielded 7h (78 mg, 61%) as a light-yellow solid. 1H NMR (DMSO-d6, 600 MHz) δ 1.82−1. 89 (m, 2H). 2.93− 2.97 (m, 2H), 2.97−3.02 (m, 2H), 3.14−3.21 (m, 4H), 4.58 (s, 2H), 5.00 (s, 2H), 5.29 (s, 2H), 6.63 (d, J = 9.1 Hz, 1H), 6.67 (d, J = 9.2 Hz, 1H), 6.99−7.04 (m, 2H), 7.11−719 (m, 1H), 7.19−7.26 (m, 2H), 7.30−7.45 (m, 5H). 13C NMR (DMSO-d6, 151 MHz) δ 28.74, 45.56, 46.07, 48.40, 51.31, 53.55, 68.23, 70.75, 108.0, 113.9, 119.6, 126.5, 126.8, 127.7, 127.9, 128.2, 128.5, 136.4, 136.6, 137.4, 141.2, 143.1, 166.3. IR (NaCl) ν 3026, 2946, 2830, 1686, 1503, 1454, 1385, 1267, 1023, 738, 695 cm−1. HPLC: (system 1) tR = 19.3 min, purity 94%. ESI-MS m/z 444.1 [M + H+]. tert-Butyl 4-[8-(Benzyloxy)-2,3-dihydrobenzo[b][1,4]dioxin-5-yl]piperazine-1-carboxylate (8d). Pd2(dba)3 (23 mg, 0.025 mmol) and rac-BINAP (46 mg, 0.07 mmol) were dissolved in toluene (9 mL) and stirred for 5 min under nitrogen atmosphere. Then NaOtBu (83 mg, 0.87 mmol), 1-boc-piperazin (290 mg, 1.56 mmol), and 8c (200 mg, 0.62 mmol) were added and the reaction mixture was stirred in a sealed microwave reaction tube at 85 °C for 1 d. After cooling down to room temperature, the reaction was diluted with EtOAc was added. The combined organic layer was washed with 0.5 M HCl, water, and brine, dried (Na2SO4), and evaporated. Purification by flash chromatography (hexanes/EtOAc 10:1 to 1:1) yielded 8d (185 mg, 70%) as orange solid. 1H NMR (DMSO-d6, 600 MHz) δ 1.48 (s, 9H), 2.89−2.94 (m, 4H), 3.53−3.63 (m, 4H), 4.29−4.33 (m, 2H), 4.33− 4.40 (m, 2H), 5.09 (s, 2H), 6.38 (d, J = 8.9 Hz, 1H), 6.45 (d, J = 8.9 Hz, 1H), 7.27−7.32 (m, 1H), 7.33−7.38 (m, 2H), 7.42−7.45 (m, 2H). 13 C NMR (DMSO-d6, 151 MHz) δ 28.61, 44.13, 51.29, 64.21, 64.39, 71.61, 79.83, 105.8, 109.2, 127.5, 128.0, 128.6, 134.9, 135.8, 137.4, 137.7, 144.5, 154.9. IR (NaCl) ν 2922, 2852, 1689, 1500, 1453, 1422, 1364, 1282, 1239, 1170, 1116, 998 cm−1. HPLC: (system 1) tR = 20.2 min, purity 92%; (system 2) tR = 16.7 min, purity 91%. ESI-MS m/z 427.1 [M + H+]. 4704



Article

7-(4-{4-[8-(Benzyloxy)quinolin-5-yl]piperazin-1-yl}butoxy)-3,4-dihydroquinolin-2(1H)-one (10e). Compound 10e was prepared as described for 10a using a solution of 7-(4-bromobutoxy)-3,4dihydroquinolin-2(1H)-one (80 mg, 0.267 mmol) and NaI (72 mg, 0.480 mmol) in acetonitrile (1.4 mL) and a solution of 9e (102 mg, 0.320 mmol) and triethylamine (62 μL, 0.480 mmol) in acetonitrile (0.7 mL). Purification by flash chromatography (CH2Cl2/MeOH 40:1 to 15:1) yielded 10e (107 mg, 74%) as a light-yellow solid. 1H NMR (DMSO-d6, 600 MHz) δ 1.59−1.68 (m, 2H), 1.71−1.81 (m, 2H), 2.37−2.46 (m, 4H), 2.52−2.73 (m, 4H), 2.78 (t, J = 7.5 Hz, 2H), 2.87−3.05 (m, 4H), 3.94 (t, J = 6.4 Hz, 2H), 5.27 (s, 2H), 6.45 (d, J = 2.5 Hz, 1H), 6.50 (dd, J = 8.2, 2.5 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 7.12 (d, J = 8.3 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 7.35 (t, J = 7.4 Hz, 1H), 7.40−7.44 (m, 2H), 7.52−7.58 (m, 3H), 8.47 (dd, J = 8.5, 1.7 Hz, 1H), 8.86 (dd, J = 4.1, 1.7 Hz, 1H), 9.97 (s, 1H). 13C NMR (DMSO-d6, 91 MHz) δ 23.34, 24.75, 27.37, 31.24, 53.12, 53.75, 58.33, 68.01, 71.04, 102.3, 108.8, 109.8, 115.7, 115.9, 121.1, 125.5, 127.3, 127.9, 128.7, 128.8, 132.1, 137.4, 138.3, 141.5, 142.5, 149.4, 151.1, 158.8, 171.7. IR (NaCl) ν 3432, 1640, 1453, 1369, 1266, 1191, 1097 cm−1. HPLC: (system 1) tR = 16.1 min, purity 99%; (system 2) tR = 13.1 min, purity 99%. ESI-MS m/z 537.6 [M + H+]. 7-(4-{4-[2,8-Bis(benzyloxy)quinolin-5-yl]-1,4-diazepan-1-yl}butoxy)-3,4-dihydroquinolin-2(1H)-one (11a). Compound 11a was prepared as described for 10a using a solution of 7-(4-bromobutoxy)3,4-dihydroquinolin-2(1H)-one (60 mg, 0.20 mmol) and NaI (55 mg, 0.36 mmol) in acetonitrile (1.0 mL) and a solution of 5h (98 mg, 0.22 mmol) and triethylamine (50 μL, 0.36 mmol) in acetonitrile (0.5 mL). Purification by flash chromatography (CH2Cl2/MeOH 40:1 to 15:1) yielded 11a (91 mg, 68%) as a yellow solid. 1H NMR (CDCl3, 360 MHz) δ 1.77−1.89 (m, 4H), 2.01−2.21 (m, 2H), 2.45−2.74 (m, 2H), 2.78−2.99 (m, 4H), 3.04−3.17 (m, 4H), 3.23 (t, J = 6.0 Hz, 2H), 3.30−3.46 (m, 2H), 3.89−4.15 (m, 2H), 5.30 (s, 2H), 5.58 (s, 2H), 6.33 (d, J = 2.4 Hz, 1H), 6.52 (dd, J = 8.3, 2.4 Hz, 1H), 6.94−6.99 (m, 2H), 7.00−7.09 (m, 2H), 7.27−7.47 (m, 6H), 7.50−7.64 (m, 4H), 7.83 (s, 1H), 8.35−8.47 (m, 1H). 13C NMR (CDCl3, 91 MHz) δ 23.21, 24.76, 27.09, 31.24, 54.18, 55.89, 56.50, 58.06, 67.79, 67.84, 71.97, 102.4, 108.8, 112.8, 113.1, 115.6, 116.1, 122.6, 127.4, 127.8, 128.0, 128.5, 128.6, 128.7, 128.9, 135.5, 137.6, 137.9, 138.3, 139.6, 150.2, 158.0, 161.3, 171.5. IR (NaCl) ν 3442, 3057, 2989, 1645, 1266, 736 cm−1. HPLC: (system 1) tR = 20.2 min, purity 97%; (system 2) tR = 17.6 min, purity 97%. ESI-MS m/z 657.4 [M + H+]. 7-(4-{4-[8-(Benzyloxy)-2-oxo-2H-chromen-5-yl]-1,4-diazepan-1yl}butoxy)-3,4-dihydroquinolin-2(1H)-one (11b). Compound 11b was prepared as described for 10a using a solution of 7-(4bromobutoxy)-3,4-dihydroquinolin-2(1H)-one (23 mg, 0.078 mmol) and NaI (17 mg, 0.117 mmol) in acetonitrile (0.8 mL) and a solution of 6g (30 mg, 0.086 mmol) and triethylamine (20 μL) in acetonitrile (0.4 mL). Purification by flash chromatography (CH2Cl2/MeOH 40:1 to 15:1) yielded 11b (20 mg, 45%) as a yellow solid. 1H NMR (CDCl3, 600 MHz) δ 1.74−1.95 (m, 4H), 2.15−2.04 (m, 2H), 2.58 (dd, J = 8.2, 6.8 Hz, 2H), 2.81−2.95 (m, 4H), 3.09−3.23 (m, 6H), 3.31−3.42 (m, 2H), 3.97 (t, J = 5.9 Hz, 2H), 5.17 (s, 2H), 6.39 (d, J = 9.8 Hz, 1H), 6.43 (d, J = 2.4 Hz, 1H), 6.50 (dd, J = 8.3, 2.4 Hz, 1H), 6.87 (d, J = 8.7 Hz, 1H), 7.01 (dd, J = 8.5, 5.0 Hz, 2H), 7.28−7.34 (m, 1H), 7.34−7.39 (m, 2H), 7.45 (dd, J = 7.9, 0.8 Hz, 2H), 8.12 (d, J = 9.8 Hz, 1H), 8.54 (s, 1H). 13C NMR (CDCl3, 91 MHz) δ 23.02, 24.69, 26.88, 26.96, 31.21, 54.22, 55.99, 58.10, 67.74, 71.83, 102.5, 108.8, 115.7, 115.7, 116.0, 116.6, 117.1, 127.6, 128.2, 128.7, 136.6, 138.4, 140.7, 142.6, 145.2, 145.4, 158.5, 160.3, 171.9. IR (NaCl) ν 3406, 1645, 1270, 1191 cm−1. HPLC: (system 1) tR = 18.0 min, purity 99%; (system 2) tR = 15.5 min, purity 99%. ESI-MS m/z 568.2 [M + H+]. 7-(4-{4-[4-Benzyl-5-(benzyloxy)-2H-benzo[b][1,4]oxazin-3(4H)one-8-yl]-1,4-diazepan-1-yl}butoxy)-3,4-dihydroquinolin-2(1H)-one (11c). Compound 11c was prepared as described for 10a, using a solution of 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)-one (31 mg, 0.10 mmol) and NaI (23 mg, 0.15 mmol) in acetonitrile (1.0 mL) and a solution of 7h (50 mg, 0.11 mmol) and triethylamine (20 μL, 0.15 mmol) in acetonitrile (0.5 mL). Purification by flash chromatography (CH2Cl2/MeOH 50:1 to 15:1) yielded 11c (46 mg, 68%) as a colorless solid. 1H NMR (CDCl3, 600 MHz) δ 1.80−

115.9, 122.1, 127.4, 127.8, 127.9, 128.5, 128.5, 128.7, 128.8, 135.5, 137.6, 138.0, 138.3, 139.7, 143.9, 150.0, 158.8, 161.2, 171.8. IR (NaCl) ν 3416, 2918, 2850, 2090, 1633, 1268 cm−1. HPLC: (system 1) tR = 19.4 min, purity 96%; (system 2) tR = 16.3 min, purity 99%. ESI-MS m/z 644.1 [M + H+]. 7-(4-{4-[8-(Benzyloxy)-2-oxo-2H-chromen-5-yl]piperazin-1-yl}butoxy)-3,4-dihydroquinolin-2(1H)-one (10b). Compound 10b was prepared as described for 10a, using a solution of 7-(4-bromobutoxy)3,4-dihydroquinolin-2(1H)-one (27 mg, 0.092 mmol) and NaI (25 mg, 0.166 mmol) in acetonitrile (0.4 mL) and a solution of 6f (34 mg, 0.101 mmol) and triethylamine (23 μL, 0.166 mmol) in acetonitrile (0.2 mL). Purification by flash chromatography (CH2Cl2/MeOH 40:1 to15:1) yielded 10b (37 mg, 74%) as a yellow solid. 1H NMR (DMSO-d6, 600 MHz) δ 1.54−1.63 (m, 2H), 1.69−1.76 (m, 2H), 2.36−2.45 (m, 4H), 2.55−2.63 (m, 4H), 2.77 (t, J = 7.5 Hz, 2H), 2.88 (s, 4H), 3.92 (t, J = 6.4 Hz, 2H), 5.20 (s, 2H), 6.43 (d, J = 2.5 Hz, 1H), 6.47 (d, J = 9.8 Hz, 1H), 6.49 (dd, J = 8.3, 2.5 Hz, 1H), 6.95 (d, J = 8.8 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 7.30 (d, J = 8.8 Hz, 1H), 7.35 (t, J = 7.3 Hz, 1H), 7.42 (dd, J = 10.3, 4.7 Hz, 2H), 7.49 (d, J = 7.2 Hz, 2H), 8.08 (d, J = 9.8 Hz, 1H), 9.96 (s, 1H). 13C NMR (DMSO-d6, 151 MHz) δ 22.73, 23.98, 26.60, 30.74, 52.90, 53.14, 57.31, 67.28, 70.43, 101.7, 107.5, 114.2, 114.3, 115.2, 115.4, 116.3, 127.8, 128.0, 128.4, 128.5, 136.7, 139.2, 140.6, 141.6, 143.7, 144.1, 157.9, 159.5, 170.2. IR (NaCl) ν 3406, 1645, 1269, 1196, 1066 cm−1. HPLC: (system 1) tR = 17.8 min, purity 99%; (system 2) tR = 15.3 min, purity 99%. ESI-MS m/z 554.4 [M + H+]. 7-(4-{4-[4-Benzyl-5-(benzyloxy)-2H-benzo[b][1,4]oxazin-3(4H)one-8-yl]piperazin-1-yl}butoxy)-3,4-dihydroquinolin-2(1H)-one (10c). Compound 10c was prepared following the protocol for 10a using a solution of 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)one (27 mg, 0.09 mmol) and NaI (24 mg, 0.16 mmol) in acetonitrile (0.9 mL) and a solution of 7g (47 mg, 0.11 mmol) and triethylamine (20 μL, 0.16 mmol) in acetonitrile (0.4 mL). Purification by flash chromatography (CH2Cl2/MeOH 40:1 to 15:1) yielded 10c (40 mg, 68%) as a colorless solid. 1H NMR (CDCl3, 600 MHz) δ 1.73−1.77 (m, 2H), 1.78−1.98 (m, 2H), 2.53−2.58 (m, 2H), 2.57−2.68 (m, 2H), 2.72−2.82 (m, 4H), 2.85−2.96 (m, 2H), 3.02−3.11 (m, 4H), 3.96 (t, J = 6.1 Hz, 2H), 4.57 (s, 2H), 4.91 (s, 2H), 5.38 (s, 2H), 6.30 (d, J = 2.4 Hz, 1H), 6.50−6.53 (m, 1H), 6.53 (d, J = 9.0 Hz, 1H), 6.61 (d, J = 9.0 Hz, 1H), 6.97−7.03 (m, 2H), 7.04 (d, J = 8.3 Hz, 1H), 7.10−7.18 (m, 3H), 7.29−7.33 (m, 2H), 7.33−7.40 (m, 3H), 7.77 (s, 1H). 13C NMR (CDCl3, 151 MHz) δ 22.96, 24.59, 27.13, 31.09, 46.73, 50.47, 53.08, 58.04, 67.76, 68.77, 71.40, 102.2, 107.7, 108.7, 114.2, 115.8, 119.9, 126.8, 126.9, 127.6, 128.2, 128.2, 128.6, 128.6, 135.5, 136.2, 137.2, 138.1, 142.6, 145.1, 158.6, 166.5, 171.7. IR (NaCl) ν 3437, 2948, 2823, 1650, 1387, 1272, 1187, 1097 cm−1. HPLC: (system 1) tR = 19.3 min, purity 99%; (system 2) tR = 16.3 min, purity 99%. ESI-MS m/z 647.5 [M + H+]. 7-(4-{4-[8-(Benzyloxy)-2,3-dihydrobenzo[b][1,4]dioxin-5-yl]piperazin-1-yl}butoxy)-3,4-dihydroquinolin-2(1H)-one (10d). Compound 10d was prepared following the protocol for 10a using a solution of 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)-one (23 mg, 0.08 mmol) and NaI (20 mg, 0.14 mmol) in acetonitrile (0.5 mL) and a solution of 8e (34 mg, 0.10 mmol) and triethylamine (19 μL, 0.14 mmol) in acetonitrile (0.3 mL). Purification by flash chromatography (CH2Cl2/MeOH 40:1 to 15:1) yielded 10d (27 mg, 64%) as a colorless solid. 1H NMR (CDCl3, 600 MHz) δ 1.68− 1.77 (m, 2H), 1.78−1.87 (m, 2H), 2.41−2.53 (m, 2H), 2.54−2.65 (m, 2H), 2.67−2.74 (m, 4H), 2.86−2.93 (m, 2H), 2.95−3.16 (m, 4H), 3.96 (t, J = 6.3 Hz, 2H), 4.30−4.32 (m, 2H), 4.32−4.35 (m, 2H), 5.09 (s, 2H), 6.27 (d, J = 2.4 Hz, 1H), 6.41 (d, J = 8.9 Hz, 1H), 6.45 (d, J = 8.9 Hz, 1H), 6.52 (dd, J = 8.3, 2.4 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 7.32−7.28 (m, 1H), 7.33−7.39 (m, 2H), 7.42−7.44 (m, 2H), 7.45 (s, 1H). 13C NMR (CDCl3, 91 MHz) δ 23.45, 24.78, 27.41, 31.27, 51.08, 53.51, 58.38, 64.20, 64.38, 68.09, 71.62, 102.3, 105.8, 108.8, 109.1, 115.9, 127.6, 128.0, 128.7, 128.9, 134.8, 137.5, 137.7, 138.2, 144.3, 158.8, 171.4. IR (NaCl) ν 3432, 2817, 1645, 1510, 1265, 1108 cm−1. HPLC: (system 1) tR = 17.9 min, purity 98%; (system 2) tR = 15.2 min, purity 98%. ESI-MS m/z 544.8 [M + H+]. 4705



Article

1611, 1598, 1478, 1280, 1250, 826, 725, 692 cm−1. HPLC: (system 1) tR = 15.4 min, purity 96%; (system 2) tR = 12.4 min, purity 96%. ESIMS m/z 467.1 [M + H+]. 7-(4-[4-(8-Hydroxy-2,3-dihydrobenzo[b][1,4]dioxin-5-yl)piperazin-1-yl]butoxy)-3,4-dihydroquinolin-2(1H)-one (12d). To a solution of 10d (73 mg, 0.12 mmol) in acetic acid/methanol (0.17 mL, 1.7 mL) under nitrogen atmosphere, a catalytic amount of 10% Pd/C was added. The flask was flushed with hydrogen, and the reaction mixture was stirred at room temperature for 4 h. After filtering over Celite and washing with MeOH, the solvent was evaporated. Purification of the crude product by column chromatography (CH2Cl2/MeOH 20:1) yielded 12d (32 mg, 61%) as a colorless solid. 1H NMR (DMSO-d6, 360 MHz) δ 1.50−1.64 (m, 2H), 1.66− 1.79 (m, 2H), 2.36−2.46 (m, 4H), 2.73−2.82 (m, 2H), 2.83−2.92 (m, 4H), 3.17 (d, J = 4.7 Hz, 2H), 3.91 (t, J = 6.3 Hz, 2H), 4.17−4.24 (m, 4H), 6.27 (s, 2H), 6.43 (d, J = 2.5 Hz, 1H), 6.48 (dd, J = 8.2, 2.5 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 8.60 (s, 1H), 9.95 (s, 1H). 13C NMR (DMSO-d6, 151 MHz) δ 21.03, 23.98, 26.58, 30.74, 48.58, 50.65, 52.98, 57.34, 63.41, 63.65, 67.26, 101.7, 106.5, 107.6, 108.9, 115.4, 128.3, 132.9, 137.4, 139.2, 157.8, 170.2. IR (NaCl) ν 3419, 2944, 1674, 1503, 1475, 1376, 1271, 1190, 1099, 988, 798, 734 cm−1. HPLC: (system 1) tR = 15.1 min, purity 96%; (system 2) tR = 12.5 min, purity 99%. ESI-MS m/z 454.1 [M + H+]. HRMS-ESI calcd 454.2336; found 454.2338 [M + H+]. 7-{4-[4-(8-Hydroxyquinolin-5-yl)piperazin-1-yl]butoxy}-3,4-dihydroquinolin-2(1H)-one·TFA (12e). Compound 12e was prepared as described for 12b, using a solution 10e (59 mg, 0.11 mmol) and TFA (0.8 mL) in toluene (0.8 mL). Purification of the crude product by preparative HPLC yielded the TFA-salt of 12e (38 mg, 62%) as an orange solid. 1H NMR (DMSO-d6, 360 MHz) δ 1.70−1.96 (m, 4H), 2.42 (dd, J = 8.3, 6.7 Hz, 2H), 2.79 (t, J = 7.5 Hz, 2H), 3.03−3.20 (m, 2H), 3.21−3.50 (m, 6H), 3.61−3.72 (m, 2H), 3.97 (t, J = 5.7 Hz, 2H), 6.45 (d, J = 2.4 Hz, 1H), 6.51 (dd, J = 8.2, 2.5 Hz, 1H), 7.07 (d, J = 8.3 Hz, 1H), 7.12 (d, J = 8.0 Hz, 1H), 7.26 (d, J = 7.5 Hz, 1H), 7.63−7.77 (m, 1H), 8.70 (d, J = 7.4 Hz, 1H), 8.90−9.20 (m, 1H), 9.72 (bs, 1H), 10.00 (s, 1H). 13C NMR (DMSO-d6, 151 MHz) δ 20.24, 23.91, 25.76, 30.66, 49.78, 51.43, 55.24, 66.61, 101.7, 107.4, 111.5, 115.6, 117.7, 121.5, 124.3, 128.3, 134.1, 138.9, 139.1, 147.3, 149.1, 157.6, 170.2. IR (NaCl) ν 3411, 3245, 2953, 2854, 1679, 1593, 1522, 1374, 1272, 1201, 1130, 1029, 832, 801, 721 cm−1. HPLC: (system 1) tR = 14.5 min, purity 99%; (system 2) tR = 11.7 min, purity 99%. ESI-MS m/z 447.4 [M + H+]. HRMS-ESI calcd 447.2391; found 447.2386 [M + H+]. 7-{4-[4-(8-Hydroxyquinolin-2(1H)-one-5-yl)-1,4-diazepan-1-yl]butoxy}-3,4-dihydroquinolin-2(1H)-one·TFA (13a). Compound 13a was prepared according to the protocol of 12c, using a solution of 11a (77 mg, 0.177 mmol) and MsOH (0.7 mL) in toluene (2 mL). Purification of the crude product by preparative HPLC yielded the TFA-salt of 13a (41 mg, 59%) as a brown solid. 1H NMR (DMSO-d6, 600 MHz) δ 1.70−1.80 (m, 2H), 1.81−1.95 (m, 2H), 2.12 (m, 2H), 2.38−2.45 (m, 2H), 2.79 (t, J = 7.5 Hz, 2H), 3.06 (t, J = 5.9 Hz, 2H), 3.23−3.31 (m, 3H), 3.31−3.42 (m, 2H), 3.46 (dd, J = 14.5, 7.0 Hz, 1H), 3.54−3.63 (m, 2H), 3.95 (t, J = 6.1 Hz, 2H), 6.44 (d, J = 2.5 Hz, 1H), 6.48−6.53 (m, 2H), 6.83 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 7.06 (d, J = 8.3 Hz, 1H), 8.16 (d, J = 9.8 Hz, 1H), 9.72 (bs, 1H), 10.01 (bs, 1H), 10.11 (bs, 1H), 10.39 (bs, 1H). 13C NMR (DMSO-d6, 151 MHz) δ 20.76, 23.97, 24.34, 25.83, 30.72, 50.72, 52.49, 54.68, 55.02, 55.39, 56.22, 66.69, 101.7, 107.4, 114.5, 114.5, 115.5, 115.7, 121.4, 128.4, 128.8, 136.6, 139.2, 140.2, 141.7, 142.8, 157.7, 161.1, 170.2. IR (NaCl) ν 3432, 1640, 1202, 1145, 1025, 986 cm−1. HPLC: (system 1) tR = 15.4 min, purity 96%; (system 2) tR = 12.4 min, purity 96%. HRMS-ESI calcd 447.2496; found 447.2491 [M + H+]. 7-{4-[4-(8-Hydroxy-2-oxo-2H-chromen-5-yl)-1,4-diazepan-1-yl]butoxy}-3,4-dihydroquinolin-2(1H)-one·TFA (13b). Compound 13b was prepared as described for 12b, using a solution of 11b (12 mg, 0.02 mmol) and TFA (0.1 mL) in toluene (0.2 mL). Purification of the crude product by preparative HPLC yielded the TFA-salt of 13b (9.5 mg, 79%) as a yellow solid. 1H NMR (DMSO-d6, 600 MHz) δ 1.70− 1.80 (m, 2H). 1.80−1.89 (m, 2H), 2.02−2.16 (m, 2H), 2.38−2.44 (m, 2H), 2.78 (t, J = 7.5 Hz, 2H), 3.05−3.12 (m, 2H), 3.16−3.26 (m, 2H), 3.42−3.54 (m, 4H), 3.94 (t, J = 6.0 Hz, 2H), 6.41−6.47 (m, 2H), 6.50

1.91 (m, 2H), 1.94−2.06 (m, 2H), 2.24−2.32 (m, 2H), 2.53−2.63 (m, 2H), 2.88 (t, J = 7.5 Hz, 2H), 2.93−3.06 (m, 2H), 3.12−3.31 (m, 6H), 3.34−3.46 (m, 2H), 3.97 (t, J = 6.0 Hz, 2H), 4.52 (s, 2H), 4.92 (s, 2H), 5.38 (s, 2H), 6.35 (d, J = 2.3 Hz, 1H), 6.48−6.55 (m, 2H), 6.57 (d, J = 9.1 Hz, 1H), 7.00−7.05 (m, 3H), 7.10−7.20 (m, 3H), 7.28− 7.32 (m, 2H), 7.32−7.40 (m, 3H), 8.00 (s, 1H). 13C NMR (CDCl3, 91 MHz) δ 22.38, 24.77, 26.95, 29.84, 31.24, 39.31, 46.93, 50.61, 53.39, 56.67, 57.70, 67.57, 68.94, 71.74, 102.4, 108.0, 108.7, 114.5, 116.2, 120.3, 127.0, 127.1, 127.7, 128.3, 128.4, 128.8, 128.9, 136.4, 137.4, 138.4, 142.1, 144.6, 158.5, 166.5, 171.5. IR (NaCl) ν 3429, 1642, 1390, 1267, 1098 cm−1. HPLC: (system 1) tR = 19.7 min, purity 96%; (system 2) tR = 16.8 min, purity 97%. ESI-MS m/z 661.3 [M + H+]. 7-{4-[4-(8-Hydroxyquinolin-2(1H)-one-5-yl)piperazin-1-yl]butoxy}-3,4-dihydroquinolin-2(1H)-one·TFA (12a). To a solution of 10a (85 mg, 0.13 mmol) in acetic acid (1.8 mL) under nitrogen atmosphere, a catalytic amount of 10% Pd/C was added. The flask was flushed with hydrogen, and the reaction mixture was stirred at room temperature for 4 h. After filtering over Celite and washing with MeOH, the solvent was evaporated. Purification of the crude product by preparative HPLC yielded the TFA-salt of 12a (38 mg, 63%) as a brown solid. 1H NMR (DMSO-d6, 600 MHz) δ 1.44−1.63 (m, 2H), 1.64−1.81 (m, 2H), 2.32−2.45 (m, 4H), 2.53−2.63 (m, 4H), 2.69− 2.80 (m, 2H), 2.80−2.89 (m, 4H), 3.91 (t, J = 6.3 Hz, 2H), 6.40−6.44 (m, 1H), 6.44−6.52 (m, 2H), 6.70 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 8.2 Hz, 1H), 7.99 (d, J = 9.8 Hz, 1H), 9.94 (s, 1H), 10.37 (s, 2H). 13C NMR (DMSO-d6, 91 MHz) δ 22.73, 23.98, 26.62, 30.73, 53.10, 53.16, 57.37, 67.30, 101.7, 107.6, 111.7, 114.5, 114.6, 115.4, 121.1, 128.3, 129.1, 136.2, 139.2, 139.7, 142.0, 157.7, 161.0, 170.2. IR (NaCl) ν 3427,2895, 2786, 1635, 1541, 1338, 1249, 1088, 889, 754, 680, 603 cm−1. HPLC: (system 1) tR = 15.3 min, purity 97%; (system 2) tR = 12.3 min, purity 96%. ESI-MS m/z 463.2 [M + H+]. HRMS-ESI calcd 463.2340; found 463.2339 [M + H+]. 7-{4-[4-(8-Hydroxy-2-oxo-2H-chromen-5-yl)piperazin-1-yl]butoxy}-3,4-dihydroquinolin-2(1H)-one (12b). To a solution of 10b (32 mg, 0.058 mmol) in toluene (0.56 mL) was added TFA (0.24 mL). The reaction mixture was stirred under reflux for 8 h. After cooling down to room temperature, the pH was adjusted to 7−8 with NaHCO3 solution and the product was extracted with CH2Cl2. The combined organic layers were dried (Na2SO4) and evaporated. Purification of the crude product by preparative HPLC yielded the TFA-salt of 12b (22 mg, 66%) as a yellow solid. 1H NMR (DMSO-d6, 360 MHz) δ 1.67−1.98 (m, 4H), 2.37−2.44 (m, 2H), 2.77 (t, J = 7.5 Hz, 2H), 2.94−3.09 (m, 2H), 3.12−3.36 (m, 6H), 3.52−3.61 (m, 2H), 3.94 (t, J = 5.7 Hz, 2H), 6.43 (d, J = 2.6 Hz, 1H), 6.45 (d, J = 9.9 Hz, 1H), 6.49 (dd, J = 8.2, 2.5 Hz, 1H), 6.94 (d, J = 8.6 Hz, 1H), 7.05 (d, J = 8.2 Hz, 1H), 7.06 (d, J = 8.6 Hz, 1H), 8.16 (d, J = 9.8 Hz, 1H), 9.67 (s, 1H), 9.98 (s, 2H). 13C NMR (DMSO-d6, 151 MHz) δ 20.78, 24.45, 26.27, 31.21, 50.46, 51.82, 55.73, 67.11, 102.2, 107.9, 115.2, 115.7, 115.8, 116.2, 118.6, 128.9, 139.7, 140.7, 141.1, 142.0, 143.4, 158.1, 160.0, 170.7. IR (NaCl) ν 3432, 2958, 2848, 1675, 1478, 1376, 1275, 1200, 1139, 1025, 994, 832, 721 cm−1. HPLC: (system 1) tR = 15.4 min, purity 97%; (system 2) tR = 12.8 min, purity 97%. ESI-MS m/z 464.3 [M + H+]. HRMS-ESI calcd 464.2180; found 464.2174 [M + H+]. 7-{4-[4-(5-Hydroxy-2H-benzo[b][1,4]oxazin-3(4H)-one-8-yl)piperazin-1-yl]butoxy}-3,4-dihydroquinolin-2(1H)-one (12c). To a suspension of 10c (9 mg, 0.01 mmol) in toluene (0.1 mL) was added MsOH (50 μL, 0.07 mmol). The reaction mixture was stirred under reflux for 3 h. After cooling down to room temperature, the solvent was removed. Purification of the crude product by flash chromatography yielded 12c (5 mg, 77%) as a colorless solid. 1H NMR (DMSOd6, 600 MHz) δ 1.69−1.79 (m, 2H), 1.79−1.89 (m, 2H), 2.38−2.47 (m, 2H), 2.76−2.85 (m, 2H), 2.89−3.00 (m, 2H), 3.11−3.18 (m, 2H), 3.18−3.24 (m, 2H), 3.30−3.35 (m, 2H), 3.48−3.61 (m, 2H), 3.94 (t, J = 6.0 Hz, 2H), 4.50 (s, 2H), 6.44−6.46 (m, 1H), 6.46−6.47 (m, 1H), 6.48−6.49 (m, 1H), 6.49−6.51 (m, 1H), 7.06 (d, J = 8.3 Hz, 1H), 9.66 (s, 1H), 9.80 (s, 1H), 9.87 (s, 1H), 10.01 (s, 1H). 13C NMR (DMSOd6, 151 MHz) δ 20.59, 24.32, 26.16, 31.07, 48.00, 51.60, 55.57, 67.00, 67.11, 102.1, 107.8, 108.8, 112.7, 116.0, 117.1, 128.7, 132.0, 137.8, 139.5, 141.7, 158.0, 164.7, 170.6. IR (NaCl) ν 3341, 3030, 2921, 2851, 4706



Article

2135, 1655, 1025, 995, 822 cm−1. HPLC: (system 1) tR = 17.8 min, purity 96%; (system 2) tR = 15.2 min, purity 92%. ESI-MS m/z 525.3 [M + H+]. 5-(4-{4-[4-Benzyl-5-(benzyloxy)-2H-benzo[b][1,4]oxazin-3(4H)one-8-yl]piperazin-1-yl}butoxy)pyrazolo[1,5-a]pyridine (14c). Compound 14c was prepared as described for 14a, using a solution 5-(4bromobutoxy)-pyrazolo[1,5-a]pyridine (100 mg, 0.37 mmol) and NaI (84 mg, 0.56 mmol) in acetonitrile (4.8 mL) and a solution of 7g (176 mg, 0.41 mmol) and triethylamine (80 μL, 0.56 mmol) in acetonitrile (0.4 mL). Purification by flash chromatography (CH2Cl2/MeOH 60:1 to 40:1) yielded 14c (170 mg, 74%) as a colorless solid. 1H NMR (DMSO-d6, 600 MHz) δ 1.55−1.66 (m, 2H), 1.74−1.81 (m, 2H), 2.38 (t, J = 7.1 Hz, 2H), 2.49−2.50 (m, 4H), 2.84−2.91 (m, 4H), 4.04 (t, J = 6.5 Hz, 2H), 4.59 (s, 2H), 5.01 (s, 2H), 5.29 (s, 2H), 6.35 (dd, J = 2.1, 0.6 Hz, 1H), 6.54 (dd, J = 7.5, 2.7 Hz, 1H), 6.62 (d, J = 9.1 Hz, 1H), 6.70 (d, J = 9.1 Hz, 1H), 6.99 (d, J = 7.2 Hz, 2H), 7.01 (d, J = 2.6 Hz, 1H), 7.15 (dd, J = 8.3, 6.3 Hz, 1H), 7.19−7.24 (m, J = 7.4 Hz, 2H), 7.32−7.40 (m, 5H), 7.86 (d, J = 2.1 Hz, 1H), 8.50 (d, J = 7.6 Hz, 1H). 13C NMR (DMSO-d6, 151 MHz) δ 22.64, 26.24, 45.62, 50.57, 52.82, 57.28, 67.77, 68.22, 70.63, 95.28, 95.61, 106.5, 107.8, 113.9, 119.4, 126.4, 126.8, 127.7, 128.0, 128.1, 128.2, 128.5, 129.6, 135.6, 136.5, 137.4, 140.6, 142.2, 142.4, 144.2, 155.0, 166.3. IR (NaCl) ν 3227, 291, 2812, 16861650, 1501, 1441, 1388, 1267, 1192, 1101, 1025, 792, 737, 698 cm−1. HPLC: (system 1) tR = 19.3 min, purity 95%; (system 2) tR = 16.5 min, purity 92%. ESI-MS m/z 618.2 [M + H+]. 5-(4-{4-[8-(Benzyloxy)-2,3-dihydrobenzo[b][1,4]dioxin-5-yl]piperazin-1-yl}butoxy)pyrazolo[1,5-a]pyridine (14d). Compound 14d was prepared as described for 14a, using a solution 5-(4bromobutoxy)-pyrazolo[1,5-a]pyridine (81 mg, 0.30 mmol) and NaI (81 mg, 0.54 mmol) in acetonitrile (4.3 mL) and a solution of 8e (118 mg, 0.36 mmol) and triethylamine (70 μL) in acetonitrile (0.4 mL). Purification by flash chromatography (CH2Cl2/MeOH 50:1) yielded 14d (118 mg, 77%) as a colorless solid. 1H NMR (DMSO-d6, 600 MHz) δ 1.54−1.71 (m, 2H), 1.73−1.81 (m, 2H), 2.35−2.40 (m, 2H), 2.84−2.93 (m, 4H), 4.05 (t, J = 6.5 Hz, 2H), 4.20−4.22 (m, 2H), 4.22−4.24 (m, 2H), 4.99 (s, 2H), 6.38−6.31 (m, 2H), 6.51 (d, J = 8.9 Hz, 1H), 6.54 (dd, J = 7.5, 2.7 Hz, 1H), 7.02 (d, J = 2.6 Hz, 1H), 7.32 (ddd, J = 7.2, 3.9, 1.3 Hz, 1H), 7.36−7.40 (m, 2H), 7.41−7.44 (m, 2H), 7.86 (d, J = 2.1 Hz, 1H), 8.50 (d, J = 7.6 Hz, 1H). 13C NMR (DMSO-d6, 91 MHz) δ 22.64, 26.23, 50.53, 52.95, 57.30, 63.43, 63.61, 67.77, 70.31, 95.26, 95.61, 105.3, 106.5, 108.3, 127.6, 127.7, 128.3, 129.5, 134.3, 135.6, 137.2, 137.4, 140.6, 142.4, 143.3, 155.0. IR (NaCl) ν 2872, 2818, 1646, 1496, 1441, 1337, 1275, 1193, 1116, 999 cm−1. HPLC: (system 1) tR = 17.9 min, purity 98%; (system 2) tR = 15.3 min, purity 95%. ESI-MS m/z 515.4 [M + H+]. 5-(4-{4-[8-(Benzyloxy)quinolin-5-yl]piperazin-1-yl}butoxy)pyrazolo[1,5-a]pyridine (14e). Compound 14e was prepared according to the protocol of 14a, using a solution 5-(4bromobutoxy)-pyrazolo[1,5-a]pyridine (80 mg, 0.30 mmol) and NaI (80 mg, 0.53 mmol) in acetonitrile (1.5 mL) and a solution of 9e (114 mg, 0.36 mmol) and triethylamine (75 μL, 0.53 mmol) in acetonitrile. Purification by flash chromatography (CH2Cl2/MeOH 60:1 to 40:1) afforded 14e (103 mg, 68%) as a yellow solid. 1H NMR (CDCl3, 600 MHz) δ 1.61−1.80 (m, 2H), 1.82−1.95 (m, 2H), 2.47−2.61 (m, 2H), 2.66−2.80 (m, 4H), 2.99−3.17 (m, 4H), 4.03 (t, J = 6.3 Hz, 2H), 5.41 (s, 2H), 6.30 (d, J = 1.7 Hz, 1H), 6.44 (dd, J = 7.5, 2.6 Hz, 1H), 6.74 (d, J = 2.5 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 7.29 (t, J = 7.4 Hz, 1H), 7.36 (t, J = 7.5 Hz, 2H), 7.43 (dd, J = 8.5, 4.2 Hz, 1H), 7.47−7.55 (m, 2H), 7.85 (d, J = 2.1 Hz, 1H), 8.28 (d, J = 7.6 Hz, 1H), 8.53 (dd, J = 8.5, 1.5 Hz, 1H), 8.97 (dd, J = 4.1, 1.7 Hz, 1H). 13 C NMR (CDCl3, 91 MHz) δ 23.57, 27.10, 53.36, 53.87, 58.33, 68.10, 71.04, 95.51, 95.55, 106.8, 109.8, 115.6, 121.0, 125.5, 127.2, 127.9, 128.7, 129.5, 132.1, 137.5, 141.2, 141.5, 142.9, 149.4, 151.1, 155.8. IR (NaCl) ν 2942, 2823, 1646, 1444, 1365, 1254, 1191, 1101 cm−1. HPLC: (system 1) tR = 15.4 min, purity 95%; (system 2) tR = 13.1 min, purity 96%. ESI-MS m/z 508.7 [M + H+]. 7-(4-{4-[2,8-Bis(benzyloxy)quinolin-5-yl]-1,4-diazepan-1-yl}butoxy)pyrazolo[1,5-a]pyridine (15a). Compound 15a was prepared as described for 14a using a solution of 5-(4-bromobutoxy)pyrazolo[1,5-a]pyridine (25 mg, 0.095 mmol) and NaI (21 mg,

(dd, J = 8.2, 2.5 Hz, 1H), 6.99 (d, J = 8.6 Hz, 1H), 7.03−7.09 (m, 2H), 8.25 (d, J = 9.8 Hz, 1H), 10.01 (s, 1H). 13C NMR (DMSO-d6, 151 MHz) δ 20.77, 23.89, 24.35, 25.81, 30.64, 52.41, 54.46, 55.36, 56.14, 66.66, 101.6, 107.4, 114.9, 115.2, 115.6, 117.3, 118.2, 128.3, 139.1, 141.1, 141.1, 142.5, 142.9, 157.6, 159.6, 170.2. IR (NaCl) ν 3427, 1640, 1267, 1196, 1147 cm−1. HPLC: (system 1) tR = 15.6 min, purity 99%. ESI-MS m/z 478.1 [M + H+]. HRMS-ESI calcd 478.2336; found 478.2337 [M + H+]. 7-{4-[4-(5-Hydroxy-2H-benzo[b][1,4]oxazin-3(4H)-one-8-yl)-1,4diazepan-1-yl]butoxy}-3,4-dihydroquinolin-2(1H)-one·TFA (13c). Compound 13c was prepared as described for 12c, using a solution of 7c (25 mg, 0.038 mmol) and MsOH (70 μL) in toluene (0.54 mL). Purification of the crude product by preparative HPLC yielded the TFA-salt of 13c (12 mg, 53%) as a light-brown solid. 1H NMR (DMSO-d6, 600 MHz) δ 1.67−1.77 (m, 2H), 1.78−1.94 (m, 2H), 2.12 (s, 2H), 2.36−2.46 (m, 2H), 2.78 (t, J = 7.5 Hz, 2H), 2.97−3.14 (m, 1H), 3.16−3.26 (m, 3H), 3.44−3.58 (m, 2H), 3.94 (t, J = 6.0 Hz, 2H), 4.50 (d, J = 3.7 Hz, 2H), 6.42 (d, J = 8.7 Hz, 1H), 6.44 (d, J = 2.4 Hz, 1H), 6.46 (d, J = 8.8 Hz, 1H), 6.50 (dd, J = 8.2, 2.5 Hz, 1H), 7.05 (d, J = 8.3 Hz, 1H), 9.50 (s, 1H), 9.67 (s, 1H), 9.82 (s, 1H), 10.00 (s, 1H). 13 C NMR (DMSO-d6, 151 MHz) δ 20.77, 23.99, 25.85, 30.74, 48.69, 49.97, 52.40, 55.15, 55.88, 66.71, 66.79, 101.7, 107.5, 108.3, 112.5, 115.7, 116.8, 128.4, 133.7, 136.9, 139.2, 140.1, 157.7, 164.5, 170.3. IR (NaCl) ν 3427, 2920, 2850, 1645, 1520, 1645, 1520, 1474, 1375, 1197, 1147, 1082 cm−1. HPLC: (system 1) tR = 15.2 min, purity 99%. ESIMS m/z 481.1 [M + H+]. HRMS-ESI calcd 481.2445; found 481.2432 [M + H+]. 5-(4-{4-[2,8-Bis(benzyloxy)quinolin-5-yl]piperazin-1-yl}butoxy)pyrazolo[1,5-a]pyridine (14a). A solution of 5-(4-bromobutoxy)pyrazolo[1,5-a]pyridine (253 mg, 0.94 mmol) and NaI (253 mg, 1.69 mmol) in acetonitrile (4.7 mL) was stirred under reflux for 0.5 h before a solution of 5g (439 mg, 1.03 mmol) and triethylamine (200 μL, 1.54 mmol) in acetonitrile (2.3 mL) was added. Stirring was continued at 80 °C for 16 h. After cooling down to room temperature, saturated NaHCO3 solution was added and the product was extracted with CH2Cl2. The combined organic layers were dried (Na2SO4) and evaporated. Purification by flash chromatography (CH2Cl2/MeOH 50:1 to 30:1) afforded 14a (382 mg, 66%) as a yellow solid. 1H NMR (DMSO-d6, 360 MHz) δ 1.59−1.69 (m, 2H), 1.75−1.87 (m, 2H), 2.40−2.47 (m, 2H), 2.57−2.76 (m, 4H), 2.87−3.01 (m, 4H), 4.07 (t, J = 6.3 Hz, 2H), 5.27 (s, 2H), 5.53 (s, 2H), 6.36 (dd, J = 2.1, 0.6 Hz, 1H), 6.54 (dd, J = 7.5, 2.6 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 2.6 Hz, 1H), 7.07 (d, J = 9.1 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 7.28−7.36 (m, 4H), 7.40 (t, J = 7.4 Hz, 2H), 7.50−7.61 (m, 4H), 7.86 (d, J = 2.1 Hz, 1H), 8.37 (d, J = 9.1 Hz, 1H), 8.50 (d, J = 7.6 Hz, 1H). 13 C NMR (DMSO-d6, 151 MHz) δ 22.71, 26.24, 53.07, 53.13, 54.89, 66.78, 67.78, 70.50, 95.29, 95.63, 106.5, 112.1, 112.6, 113.1, 121.1, 127.2, 127.6, 127.7, 128.2, 128.3, 128.6, 129.6, 135.5, 137.2, 137.8, 138.6, 140.6, 142.4, 155.0, 160.2. IR (NaCl) ν 3442, 3050, 2951, 2822, 1648, 1610, 1445, 1409, 1264, 1191, 1009, 897 cm−1. HPLC: (system 1) tR = 20.1 min, purity 99%; (system 2) tR = 17.6 min, purity 99%. ESI-MS m/z 615.0 [M + H+]. 5-(4-{4-[8-(Benzyloxy)-2-oxo-2H-chromen-5-yl]piperazin-1-yl}butoxy)pyrazolo[1,5-a]pyridine (14b). Compound 14b was prepared as described for 14a, using a solution 5-(4-bromobutoxy)-pyrazolo[1,5-a]pyridine (33 mg, 0.122 mmol) and NaI (33 mg, 0.220 mmol) in acetonitrile (0.6 mL) and a solution of 6f (45 mg, 0.134 mmol) and triethylamine (30 μL, 0.220 mmol) in acetonitrile (0.2 mL). Purification by flash chromatography (CH2Cl2/MeOH 40:1) yielded 14b (42 mg, 65%) as a colorless solid. 1H NMR (DMSO-d6, 600 MHz) δ 1.55−1.70 (m, 2H), 1.70−1.92 (m, 2H), 2.39−2.47 (m, 2H), 2.55−2.74 (m, 4H), 2.80−2.97 (m, 4H), 4.06 (t, J = 6.5 Hz, 2H), 5.20 (s, 2H), 6.33−6.40 (m, 1H), 6.47 (d, J = 9.8 Hz, 1H), 6.54 (dd, J = 7.5, 2.7 Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H), 7.02 (d, J = 2.6 Hz, 1H), 7.29 (d, J = 8.8 Hz, 1H), 7.35 (t, J = 7.3 Hz, 1H), 7.42 (dd, J = 10.3, 4.7 Hz, 2H), 7.49 (d, J = 7.3 Hz, 2H), 7.86 (d, J = 2.1 Hz, 1H), 8.08 (d, J = 9.8 Hz, 1H), 8.50 (d, J = 7.5 Hz, 1H). 13C NMR (DMSO-d6, 151 MHz) δ 22.57, 26.14, 52.80, 53.05, 57.10, 67.67, 70.34, 95.20, 95.53, 106.4, 114.1, 114.2, 115.1, 116.2, 127.7, 127.9, 128.7, 130.0, 136.6, 140.2, 140.5, 141.6, 142.3, 143.6, 144.0, 155.0, 159.4. IR (NaCl) ν 2255, 4707



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(system 2) tR = 12.1 min, purity 97%. ESI-MS m/z 438.1 [M + H+]. HRMS-ESI calcd 438.2136; found 438.2135 [M + H+]. 5-(4-[4-(8-Hydroxy-2,3-dihydrobenzo[b][1,4]dioxin-5-yl)piperazin-1-yl]butoxy)pyrazolo[1,5-a]pyridine (16d). Compound 16d was prepared as described for 12a, using a solution of 14d (112 mg, 0.22 mmol) and a catalytic amount of 10% Pd/C in AcOH/ MeOH 1:1 (2.2 mL). Purification of the crude product by flash chromatography (CH2Cl2/MeOH 60:1 to 30:1) yielded 16d (74 mg, 79%) as a colorless solid. 1H NMR (DMSO-d6, 600 MHz) δ 1.56− 1.64 (m, 2H), 1.75−1.82 (m, 2H), 2.37 (t, J = 7.2 Hz, 2H), 2.46−2.49 (m, 4H), 2.77−2.94 (m, 4H), 4.04 (t, J = 6.5 Hz, 2H), 4.19 (dd, J = 3.4, 1.6 Hz, 2H), 4.21 (dd, J = 3.5, 1.5 Hz, 2H), 6.26 (s, 2H), 6.35 (dd, J = 2.2, 0.8 Hz, 1H), 6.54 (dd, J = 7.5, 2.6 Hz, 1H), 7.02 (d, J = 2.6 Hz, 1H), 7.86 (d, J = 2.1 Hz, 1H), 8.49 (d, J = 7.6 Hz, 1H), 8.62 (s, 1H). 13 C NMR (DMSO-d6, 151 MHz) δ 22.69, 26.27, 50.77, 53.07, 57.38, 63.44, 63.67, 67.81, 95.31, 95.63, 106.5, 106.5, 108.9, 129.6, 132.9, 133.7, 137.4, 140.6, 141.8, 142.4, 155.1. IR (NaCl) ν 3406, 2952, 2879, 2825, 1645, 1445, 1343, 1267, 1189, 1093, 990 cm−1. HPLC: (system 1) tR = 15.0 min, purity 93%; (system 2) tR = 12.5 min, purity 94%. HRMS-ESI calcd 425.2184; found 425.2188 [M + H+]. 5-{4-[4-(8-Hydroxyquinolin-5-yl)piperazin-1-yl]butoxy}pyrazolo[1,5-a]pyridine·TFA (16e). Compound 16e was prepared according to the protocol of 12b, using a solution of 14e (70 mg, 0.14 mmol) and TFA (1.0 mL) in toluene (1.0 mL). Purification of the crude product by preparative HPLC yielded the TFA-salt of 16e (48 mg, 66%) as a yellow solid. 1H NMR (DMSO-d6, 360 MHz) δ 1.80−1.95 (m, 4H), 3.00−3.19 (m, 2H), 3.26−3.47 (m, 6H), 3.71−3.57 (m, 2H), 4.09 (t, J = 5.7 Hz, 2H), 6.37 (d, J = 1.7 Hz, 1H), 6.54 (dd, J = 7.5, 2.7 Hz, 1H), 7.03 (d, J = 2.6 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 7.29 (d, J = 8.2 Hz, 1H), 7.76 (dd, J = 8.0, 4.4 Hz, 1H), 7.86 (d, J = 2.1 Hz, 1H), 8.52 (d, J = 7.6 Hz, 1H), 8.80 (d, J = 8.4 Hz, 1H), 8.95 (d, J = 3.5 Hz, 1H), 9.91 (bs, 1H). 13C NMR (DMSO-d6, 91 MHz) δ 20.81, 26.02, 50.38, 52.04, 55.77, 67.70, 95.91, 96.29, 106.9, 112.8, 118.8, 122.2, 125.1, 130.2, 136.3, 141.1, 143.0, 147.3, 155.4, 158.9. IR (NaCl) ν 3401, 3096, 2921, 2851, 1680, 1445, 1385, 1198, 1132, 1027, 837, 799, 723 cm−1. HPLC: (system 1) tR = 14.3 min, purity 98%; (system 2) tR = 11.6 min, purity 99%. HRMS-ESI calcd 418.2238; found 418.2229 [M + H+]. 5-{4-[4-(8-Hydroxyquinolin-2(1H)-one-5-yl)-1,4-diazepan-1-yl]butoxy}pyrazolo[1,5-a]pyridine·TFA (17a). Compound 17a was prepared as described for 12c, using a solution of 15a (16 mg, 0.025 mmol) and MsOH (0.02 mL) in toluene (0.4 mL). Purification of the crude product by preparative HPLC yielded the TFA-salt of 17a (6.7 mg, 48%) as a light-brown solid. 1H NMR (DMSO-d6, 360 MHz) δ 1.75−1.96 (m, 4H), 2.11−2.18 (m, 2H), 3.07 (t, J = 5.7 Hz, 2H), 3.22−3.33 (m, 2H), 3.55−3.66 (m, 2H), 4.08 (t, J = 5.8 Hz, 2H), 6.38 (d, J = 1.3 Hz, 1H), 6.50 (d, J = 9.8 Hz, 1H), 6.54 (dd, J = 7.5, 2.6 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 7.04 (d, J = 2.4 Hz, 1H), 7.87 (d, J = 1.8 Hz, 1H), 8.17 (d, J = 9.8 Hz, 1H), 8.52 (d, J = 7.6 Hz, 1H), 10.04 (s, 1H), 10.14 (s, 1H), 10.38 (s, 1H). 13C NMR (DMSO-d6, 151 MHz) δ 21.17, 24.75, 26.00, 51.15, 52.92, 55.12, 55.90, 56.60, 67.70, 95.85, 96.21, 106.9, 115.0, 115.0, 116.0, 116.7, 121.8, 129.2, 130.1, 137.1, 140.7, 141.0, 142.9, 143.3, 155.3, 158.3, 158.7, 161.6. IR (NaCl) ν 3427, 2927, 2854, 1653, 1560, 1445, 1193 cm−1. HPLC: (system 1) tR = 15.4 min, purity 99%; (system 2) tR = 12.5 min, purity 96%. ESI-MS m/z 448.2 [M + H+]. HRMS-ESI calcd 448.2330; found 448.2335 [M + H+]. N-(3-(4-(2,8-Bis(benzyloxy)quinolin-5-yl)-1,4-diazepan-1-yl)propyl)-4-(3-(1-butyl-1H-1,2,3-triazol-4-yl)propoxy)-3-methoxybenzamide (18a). Compound 5h (25 mg, 0.06 mmol) and 4-(3-(1-butyl1H-1,2,3-triazol-4-yl)propoxy)-N-(3-oxopropyl)-3-methoxybenzamide (44 mg, 0.11 mmol) were dissolved in dichloroethane (2.8 mL), and NaBH(OAc)3 (48 mg, 0.23 mmol) was added. The mixture was stirred at room temperature for 6 h. After addition of 1 M NaOH, the product was extracted with CH2Cl2. The combined organic layers were dried (Na2SO4) and evaporated. After column chromatography (CH2Cl2/ MeOH 20:1) 18a was obtained as crude yellow solid. 18a was used in further reactions without a final purification step. ESI-MS m/z 812.4 [M + H+] N-(4-(4-(2,8-Bis(benzyloxy)quinolin-5-yl)-1,4-diazepan-1-yl)butyl)-4-(3-(1-butyl-1H-1,2,3-triazol-4-yl)propoxy)-3-methoxyben-

0.142 mmol) in acetonitrile (0.95 mL) and a solution of 5h (176 mg, 0.409 mmol) and triethylamine (80 μL, 0.558 mmol) in acetonitrile (0.4 mL). Purification by flash chromatography (CH2Cl2/MeOH 60:1 to 40:1) yielded 15a (170 mg,74%) as a colorless solid. 1H NMR (CDCl3, 360 MHz) δ 1.85−1.98 (m, 4H), 2.07−2.19 (m, 2H), 2.77− 2.94 (m, 2H), 3.06−3.19 (m, 4H), 3.24 (t, J = 6.0 Hz, 2H), 3.39 (m, 2H), 4.03 (t, J = 5.7 Hz, 2H), 5.30 (s, 2H), 5.58 (s, 2H), 6.30 (dd, J = 2.2, 0.7 Hz, 1H), 6.43 (dd, J = 7.6, 2.6 Hz, 1H), 6.73 (d, J = 2.5 Hz, 1H), 6.90−7.00 (m, 2H), 7.04 (d, J = 8.4 Hz, 1H), 7.28−7.43 (m, 6H), 7.50−7.62 (m, 4H), 7.85 (d, J = 2.2 Hz, 1H), 8.28 (d, J = 7.6 Hz, 1H), 8.40 (d, J = 9.1 Hz, 1H). 13C NMR (CDCl3, 91 MHz) δ 22.07, 26.64, 55.85, 56.55, 57.78, 67.38, 67.91, 71.80, 95.71, 95.81, 106.6, 112.8, 113.2, 116.3, 122.5, 127.4, 127.9, 128.0, 128.5, 128.6, 128.7, 129.6, 135.0, 137.4, 137.7, 139.5, 141.1, 143.0, 150.7, 155.4, 161.3. IR (NaCl) ν 3031, 2927, 2849, 2599, 1646, 1443, 1410, 1265, 1191, 1095, 1002 cm−1. HPLC: (system 1) tR = 20.3 min, purity 83%; (system 2) tR = 17.9 min, purity 83%. ESI-MS m/z 628.2 [M + H+]. 5-{4-[4-(8-Hydroxyquinolin-2(1H)-one-5-yl)piperazin-1-yl]butoxy}pyrazolo[1,5-a]pyridine (16a). Compound 16a was prepared as described for 12a, using a solution of 14a (378 mg, 0.62 mmol) and a catalytic amount of 10% Pd/C in AcOH/MeOH 1:1 (8.8 mL). Purification of the crude product flash chromatography (CH2Cl2/ MeOH 25:1 + 1% NH3) yielded 16a (185 mg, 70%) as a yellow solid. 1 H NMR (DMSO-d6, 600 MHz) δ 1.57−1.71 (m, 2H), 1.76−1.86 (m, 2H), 2.43 (t, J = 7.2 Hz, 2H), 2.55−2.64 (m, 4H), 2.82−2.90 (m, 4H), 4.06 (t, J = 6.5 Hz, 2H), 6.36 (d, J = 1.5 Hz, 1H), 6.48 (d, J = 9.8 Hz, 1H), 6.54 (dd, J = 7.5, 2.7 Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 8.3 Hz, 1H), 7.02 (d, J = 2.6 Hz, 1H), 7.86 (d, J = 2.1 Hz, 1H), 8.00 (d, J = 9.8 Hz, 1H) 8.50 (d, J = 7.5 Hz, 1H), 10.21 (s, 2H). 13C NMR (DMSO-d6, 101 MHz) δ 22.60, 26.16, 53.02, 53.09, 57.18, 67.68, 95.19, 95.50, 106.4, 111.6, 114.3, 114.5, 121.0, 129.0, 129.5, 136.1, 139.7, 140.5, 141.9, 142.3, 155.0, 161.0. IR (NaCl) ν 3421, 2949, 2819, 1643, 1485, 1442, 1369, 1345, 1442, 1276, 1190, 1145 cm−1. HPLC: (system 1) tR = 15.1 min, purity 99%; (system 2) tR = 12.3 min, purity 97%. HRMS-ESI calcd 434.2187; found 434.2183 [M + H+]. 5-{4-[4-(8-Hydroxy-2-oxo-2H-chromen-5-yl)piperazin-1-yl]butoxy}pyrazolo[1,5-a]pyridine·TFA (16b). Compound 16b was prepared as described for 12b, using a solution of 14b (35 mg, 0.067 mmol) and TFA (0.29 mL) in toluene (0.67 mL). Purification of the crude product by preparative HPLC yielded the TFA-salt of 16b (21 mg, 57%) as a yellow solid. 1H NMR (DMSO-d6, 600 MHz) δ 1.79−1.91 (m, 4H), 3.0−3.12 (m, 2H), 3.12−3.39 (m, 6H), 3.53−3.69 (m, 2H), 4.09 (t, J = 5.5 Hz, 2H), 6.38 (d, J = 1.8 Hz, 1H), 6.47 (d, J = 9.8 Hz, 1H), 6.55 (dd, J = 7.5, 2.7 Hz, 1H), 6.95 (d, J = 8.6 Hz, 1H), 7.04 (d, J = 2.6 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H), 7.88 (d, J = 2.1 Hz, 1H), 8.18 (d, J = 9.8 Hz, 1H), 8.53 (d, J = 7.6 Hz, 1H), 9.71 (s, 1H), 10. 00 (s, 1H). 13C NMR (DMSO-d6, 151 MHz) δ 20.28, 25.47, 49.99, 51.37, 55.20, 67.16, 95.40, 95.74, 106.4, 114.8, 115.3, 115.3, 118.1, 129.7, 140.2, 140.5, 140.6, 141.6, 142.5, 142.9, 154.9, 159.5. IR (NaCl) ν 3406, 3077, 2941, 2845, 1715, 1648, 1439, 1198, 1128, 1047, 1024, 826, 718, 668 cm−1. HPLC: (system 1) tR = 15.3 min, purity 99%; (system 2) tR = 12.9 min, purity 99%. HRMS-ESI calcd 435.2027; found 435.2031 [M + H+]. 5-{4-[4-(5-Hydroxy-2H-benzo[b][1,4]oxazin-3(4H)-one-8-yl)piperazin-1-yl]butoxy}pyrazolo[1,5-a]pyridine·TFA (16c). Compound 16c was prepared as described for 12c, using a solution 14c (99 mg, 0.16 mmol) and MsOH (0.31 mL) in toluene (2.3 mL). Purification of the crude product by preparative HPLC yielded the TFA-salt of 16c (24 mg, 28%) as colorless solid. 1H NMR (DMSO-d6, 600 MHz) δ 1.73−1.94 (m, 4H), 2.91 (t, J = 11.8 Hz, 2H), 3.13−3.21 (m, 2H), 3.21−3.27 (m, 2H), 3.33−3.42 (m, 2H), 3.54−3.61 (m, 2H), 4.07 (t, J = 5.9 Hz, 2H), 4.51 (s, 2H), 6.38 (dd, J = 2.1, 0.7 Hz, 1H), 6.46 (d, J = 8.7 Hz, 1H), 6.50 (d, J = 8.8 Hz, 1H), 6.55 (dd, J = 7.5, 2.7 Hz, 1H), 7.03 (d, J = 2.6 Hz, 1H), 7.88 (d, J = 2.1 Hz, 1H), 8.53 (d, J = 7.6 Hz, 1H), 9.64−9.76 (m, 2H), 9.87 (s, 1H). 13C NMR (DMSOd6, 91 MHz) δ 20.18, 25.38, 47.63, 51.23, 55.13, 66.69, 67.08, 95.29, 95.67, 106.3, 108.3, 112.4, 116.7, 129.6, 131.6, 137.4, 140.4, 141.3, 142.4, 154.8, 164.2. IR (NaCl) ν 3432, 2964, 2925, 2851, 1650, 1261, 1193, 1075 cm−1. HPLC: (system 1) tR = 14.7 min, purity 95%; 4708



Article

MS m/z 646.3 [M + H+]. HRMS-ESI calcd 323.6892; found 323.6890 [M/2 + 2H+]. Membrane Preparation. Membranes were obtained using a previously described protocol.62 Briefly, HEK293T cells were grown to a confluence of 70% and transiently transfected with the receptor of interest with or without cotransfection of a PTX insensitive Gαo1 subunit using Mirus TransIT-293 transfection reagent (MoBiTec, Goettingen, Germany), CaHPO4 precipitation, or a solution of linear polyethylenimine in PBS as described previously.28,29 After 24 h of incubation with the transfection complex, the medium was replaced. 48 h after transfection, cells were washed with ice-cold phosphate buffered saline (PBS, pH 7.4) and detached with homogenate buffer (10 mM Tris-HCl, 0.5 mM EDTA, 5.4 mM KCl, 140 mM NaCl, pH 7.4). After centrifugation (8 min, 220g), the pellet was resuspended in 10 mL of ice-cold homogenate buffer and cells were lysed with an ultraturrax. After ultracentrifugation (30 min 50000g), membranes were resuspended in binding buffer (50 mM Tris-HCl, 1 mM EDTA, 5 mM MgCl2, 100 μg/mL bacitracin, 5 μg/mL soybean trypsin inhibitor, pH 7.4) and homogenized with a glass−Teflon homogenizer at 4 °C. Membrane preparations were shock-frozen in liquid nitrogen and stored at −80 °C. The protein concentration was determined with the method of Lowry63 and bovine serum albumin as standard. Membranes from stably transfected CHO cells were obtained analogously. Competition Binding Experiments. Affinities of the test compounds toward the human D1R, D2LR, D2SR, D3R, D4R, D5R, 5HT2AR, and porcine α1-AR and 5-HT1AR were determined as described previously.64 In brief, membranes containing human D2LR, D2SR, D3R, or D4R obtained from CHO cells stably expressing the corresponding receptors were used together with the radioligand [3H]spiperone (specific activity of 73 Ci/mmol, PerkinElmer, Rodgau, Germany) at a final concentration of 0.10−0.20 nM (KD 0.035−0.10, Bmax 610−1100 fmol/mg, protein concentration 4−6 μg/test tube), 0.15−0.20 nM (KD 0.032−0.11, Bmax 1500−8500 fmol/mg, protein concentration 1−4 μg/test tube), 0.20−0.50 nM (KD 0.08−0.55, Bmax 3300−8500 fmol/mg, protein concentration 1−2 μg/test tube), and 0.20−0.50 nM (KD 0.14−0.35, Bmax 600−4500 fmol/mg, protein concentration 5−14 μg/test tube) for D2LR, D2SR, D3R, and D4R, respectively. Competition binding experiments with the human D1R, D5R, and 5-HT2AR were performed in an analogous manner with membranes from transiently transfected HEK293T cells together with [3H]SCH23390 (80 Ci/mmol, Biotrend, Cologne, Germany, final concentration 0.20−0.50 nM, KD 0.14−0.45 nM, Bmax 2200−8900 fmol/mg, protein concentration 2−4 μg/test tube for D1R and 0.40− 0.60 nM, KD 0.34−0.71 nM, Bmax 450−2750 fmol/mg, 5−10 μg/test tube for D5R) or [3H]ketanserin (47 Ci/mmol, PerkinElmer, final concentration 0.30−0.50 nM, KD 0.20−0.40 nM, Bmax 1400−2400 fmol/mg, protein concentration 3−6 μg/test tube for 5-HT2AR). Binding studies with α1R or 5-HT1AR were carried out as described previously with homogenates derived from porcine cerebral cortex64 in the presence of the radioligands [3H]prazosin (84 Ci/mmol, PerkinElmer, 0.15−0.20 nM final concentration, KD 0.060−0.170 nM, Bmax 150−350 fmol/mg, protein concentration 20−40 μg/test tube for α1R) or [3H]WAY100635 (80 Ci/mmol, Biotrend, 0.20−0.35 nM final concentration, KD 0.035−0.065 nM, Bmax 40−120 fmol/mg, protein concentration 60−80 μg/test tube for 5-HT1AR). Unspecific binding was determined in the presence of haloperidol (10 μM for D1R-D5R), WAY100635 (10 μM for 5-HT1AR), ketanserin (10 μM for 5-HT2AR), or prazosin (10 μM for α1R).64 Resulting competition curves were analyzed by nonlinear regression using the algorithms for one-site competition of PRISM 6.0 (GraphPad, San Diego, CA). β-Arrestin-2 Recruitment. The PathHunter assay (DiscoverX, Birmingham, U.K.) was employed to determine β-arrestin-2 recruitment at D2SR as described previously.28 In brief, (EA)-β-arrestin-2HEK293 cells were transiently transfected with D2SR−ARMS2−PK2 employing Mirus TransIT-293 (MoBiTec, Göttingen, Germany). Experiments in the presence of GRK2 were performed analogously, using the cDNAs of D2SR−ARMS2−PK2 and hGRK2 in a 2:1 ratio for transfection. Then 24 h after transfection, cells were detached with Versene (Life Technologies, Darmstadt, Germany), seeded into white

zamide (18b). Compound 18b was prepared as described for 18a, using a solution of 5h (10 mg, 0.02 mmol) and 4-[3-(1-butyl-1H-1,2,3triazol-4-yl)propoxy]-N-(4-oxobutyl)-3-methoxybenzamide38,61 (18 mg, 0.04 mmol) and NaBH(OAc)3 (19 mg, 0.09 mmol) in DCE (1.1 mL). Purification of the crude product by flash chromatography (CH2Cl2/MeOH 40:1) yielded 18b (15 mg, 80%) as light-yellow solid. 1H NMR (DMSO-d6, 600 MHz) δ 0.87 (t, J = 7.4 Hz, 3H), 1.22 (dq, J = 14.7, 7.3 Hz, 2H), 1.58 (dt, J = 14.1, 6.9 Hz, 2H), 1.69−1.80 (m, 4H), 1.98−2.11 (m, 2H), 2.15 (s, 2H), 2.77 (t, J = 7.5 Hz, 2H), 3.14 (t, J = 5.6 Hz, 2H), 3.24 (s, 2H), 3.29−3.33 (m, 2H), 3.41−3.52 (m, 4H), 3.59 (s, 2H), 3.81 (s, 3H), 4.05 (t, J = 6.3 Hz, 2H), 4.29 (t, J = 7.0 Hz, 2H), 5.29 (s, 2H), 5.53 (s, 2H), 6.99 (d, J = 8.9 Hz, 1H), 7.10 (t, J = 8.9 Hz, 2H), 7.21 (d, J = 8.5 Hz, 1H), 7.27−7.36 (m, 3H), 7.41 (dd, J = 17.7, 10.2 Hz, 2H), 7.44−7.51 (m, 2H), 7.53−7.59 (m, 3H), 7.90 (s, 1H), 8.45 (t, J = 5.6 Hz, 1H), 8.50 (d, J = 9.0 Hz, 1H), 10.06 (s, 1H). 13C NMR (DMSO-d6, 91 MHz) δ 13.23, 19.04, 21.30, 21.56, 24.32, 26.43, 28.44, 30.62, 31.66, 38.40, 48.83, 50.64, 52.50, 54.57, 55.17, 55.63, 56.22, 66.79, 67.48, 70.44, 110.9, 112.0, 112.4, 112.5, 115.8, 120.3, 121.8, 121.8, 126.8, 127.2, 127.6, 127.7, 128.2, 128.3, 128.5, 135.9, 137.2, 137.7, 138.4, 143.9, 146.0, 148.4, 149.5, 150.5, 157.8, 158.1, 160.3, 165.7. IR (NaCl) ν 3421, 2948, 2864, 1681, 1506, 1471, 1410, 1320, 1267, 1201, 1130, 1026 cm−1. HPLC: (system 1) tR = 20.6 min, purity 94%; (system 2) tR = 18.0 min, purity 95%. ESI-MS m/z 826.4 [M + H+] N-(3-(4-(8-Hydroxyquinolin-2(1H)-one-5-yl)-1,4-diazepan-1-yl)propyl)-4-(3-(1-butyl-1H-1,2,3-triazol-4-yl)propoxy)-3-methoxybenzamide (19a). To a solution of 18a (25 mg, 0.03 mmol) in toluene (0.44 mL) was added MsOH (20 μL), and the mixture was stirred at 80 °C for 1 h. After cooling down to room temperature, the solvent was evaporated. The residue was purified by preparative HPLC to yield the TFA salt of 19a (10 mg, 43%) as a yellow solid. 1H NMR (DMSO-d6, 600 MHz) δ 0.88 (t, J = 7.4 Hz, 3H), 1.18−1.28 (m, 2H), 1.69−1.83 (m, 2H), 1.92−2.02 (m, 2H), 2.0−2.09 (m, 2H), 2.09−2.16 (m, 2H), 2.78 (t, J = 7.6 Hz, 2H), 3.06 (t, J = 5.9 Hz, 2H), 3.20−3.31 (m, 3H), 3.32−3.43 (m, 4H), 3.43−3.52 (m, 2H), 3.54−3.65 (m, 3H), 3.82 (s, 3H), 4.06 (t, J = 6.4 Hz, 2H), 4.29 (t, J = 7.1 Hz, 2H), 6.49 (d, J = 9.8 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 7.01 (d, J = 8.9 Hz, 1H), 7.43−7.49 (m, 2H), 7.91 (s, 1H), 8.18 (d, J = 9.8 Hz, 1H), 8.56 (t, J = 5.7 Hz, 1H), 9.82 (s, 1H), 10.12 (s, 1H), 10.39 (s, 1H). 13C NMR (DMSO-d6, 151 MHz) δ 13.19, 18.98, 21.49, 24.34, 24.43, 28.36, 31.61, 36.34, 48.77, 50.68, 52.42, 54.53, 54.61, 55.30, 55.56, 67.40, 110.8, 111.8, 114.5, 115.4, 115.6, 117.6, 120.4, 121.3, 121.7, 126.4, 128.7, 136.5, 140.2, 142.7, 145.9, 148.3, 150.5, 157.8, 158.0, 161.0, 166.0. IR (NaCl) ν 3437, 2958, 2854, 1650, 1510, 1428, 1374, 1327, 1277, 1202, 1142, 1019, 843, 802, 720 cm−1. HPLC: (system 1) tR = 16.7 min, purity >99%. ESI-MS m/z 623.3 [M + H+]. HRMS-ESI calcd 316.6815; found 316.6815 [M + H+]. N-(4-(4-(8-Hydroxyquinolin-2(1H)-one-5-yl)-1,4-diazepan-1-yl)butyl)-4-(3-(1-butyl-1H-1,2,3-triazol-4-yl)propoxy)-3-methoxybenzamide (19b). To a solution of 18b (19 mg, 0.02 mmol) in TFA/ toluene (1:1, 0.4 mL) two drops of MsOH were added before the mixture was stirred at 80 °C for 1 h. After cooling down to room temperature, the solvent was evaporated and the crude residue was purified by preparative HPLC to yield the TFA-salt 19b (11 mg, 74%) as a yellow solid. 1H NMR (DMSO-d6, 600 MHz) δ 0.87 (t, J = 7.4 Hz, 3H), 1.14−1.35 (m, 2H), 1.66−1.51 (m, 2H), 1.84−1.69 (m, 4H), 2.10−2.02 (m, 2H), 2.21−2.10 (m, 2H), 2.77 (t, J = 7.5 Hz, 2H), 3.09−2.99 (m, 2H), 3.24−3.19 (m, 2H), 3.32−3.24 (m, 4H), 3.60− 3.51 (m, 2H), 3.82 (s, 3H), 4.05 (t, J = 6.3 Hz, 2H), 4.29 (t, J = 7.1 Hz, 2H), 6.49 (d, J = 9.8 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.99 (d, J = 9.0 Hz, 1H), 7.51−7.43 (m, 2H), 7.91 (s, 1H), 8.17 (d, J = 9.5 Hz, 1H), 8.48 (t, J = 5.6 Hz, 1H), 10.23 (s, 1H), 10.33 (s, 1H), 10.42 (s, 1H). 13C NMR (DMSO-d6, 151 MHz) δ 13.19, 18.98, 21.13, 21.50, 24.11, 26.33, 28.37, 31.60, 38.30, 48.48, 48.76, 50.53, 52.27, 54.45, 55.36, 55.56, 56.03, 67.38, 110.8, 111.8, 114.4, 114.5, 115.4, 116.2, 120.3, 121.2, 121.7, 126.7, 128.7, 136.6, 140.2, 142.7, 145.9, 148.3, 150.4, 157.8, 158.0, 161.1, 165.6. IR (NaCl) ν 3421, 1644, 1562, 1490, 1421, 1371, 1275, 1205, 1144, 1025, 982, 802, 719. cm−1. HPLC: (system 1) tR = 16.7 min, purity >98%. ESI4709



Article

384-well plates (Greiner, Frickenhausen, Germany) in assay medium at a density of 5000 cells/well, and maintained for 24 h at 37 °C, 5% CO2. After incubation with the test compounds dissolved in PBS for 5 h at 37 °C, the detection mix was added and incubation was continued for 60 min at room temperature. Chemiluminescence was determined using the CLARIOstar plate reader (BMG LabTech, Ortenberg, Germany). Then 3−8 experiments per compound were performed with each concentration in duplicate. All responses were normalized to the effect of 10 μM quinpirole (100%) and buffer conditions (0%) and analyzed using the algorithms for nonlinear regression in PRISM 6.0. [35S]GTPγS Binding Experiments. [35S]GTPγS incorporation assays were performed in analogy to previously described protocols.28 Membranes from HEK293T cells transiently transfected with D2SR and a pertussis toxin insensitive variant of Gαo1 (protein concentration 15 μg/mL, final volume 200 μL, Bmax 9900 ± 140 fmol/mg) were incubated with 10 μM GDP and the test compounds in assay buffer (20 mM HEPES, 10 mM MgCl2·6H2O, 100 mM NaCl, 70 mg/L saponin) for 30 min at 37 °C. After the addition of [35S]GTPγS (0.1 nM final concentration, specific activity 1250 Ci/mmol, PerkinElmer), incubation was continued for 30 min at 37 °C before the reaction was terminated by filtration through Whatman GF/B filters. Bound radioactivity was determined as described previously.62 Then 4−6 experiments per compound were performed with each concentration (0.1 pM to 100 μM) in triplicate. Responses were normalized to the maximum effect of the full agonist quinpirole (100%) and buffer conditions (0%). Analysis of dose response curves was performed using the algorithms for nonlinear regression in PRISM 6.0. Operational Model of Agonism. Quantification of ligand bias was performed using the operational model of agonism65 following recently published protocols.66,67 Using the algorithms implemented in PRISM6.0 (Graphpad), transduction ratios (τ/KA) were obtained in their logarithmic form. Thus, normalized data from the functional experiments was analyzed using the following equation:

n

( ) [X ] τ KA

n

T1/2(min) =

τ KA

hERG Inhibition. Inhibition of hERG (human-ether-à-go-go related gene) tail currents was examined using automated whole cell patch clamp and CHO cells stably transfected with the human KCNH2 gene at B’SYS Analytics, Switzerland. All experiments were conducted at room temperature using a bath solution containing 140 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM D-glucose adjusted to pH 7.4 with NaOH. A solution of 130 mM KCl, 2 mM CaCl2, 4 mM MgCl2, 4 mM Na2-ATP, 10 mM HEPES, and 5 mM EGTA adjusted to pH 7.2 with KOH was used as intracellular solution. Outward tail currents were measured upon depolarization of the cell membrane from a holding potential of −80 mV to +20 mV for 2 s (activation of channels) and upon subsequent repolarization to −40 mV for 3 s. After a waiting time of 5 s, the next pulse followed. The application protocol consisted of five periods, during which vehicle (DMSO 0.5%) and 13a were applied in increasing concentrations. The voltage protocol was executed 20 times during each application period. As reference, the selective IKr inhibitor E-4031 (Sigma, Germany) was employed (final concentration 100 nM). The tail current amplitudes at the end of each period were used in further evaluation steps. The amount of current block was calculated as percentage of control, and the data was analyzed with the following equation to generate a dose−response curve and to determine an IC50, when n is the hill slope and [X] the concentration of the test compound:

n

[X ] KA

n

with basal being the response of a the system in absence of an agonist, Em the maximal response, [X] the concentration of the agonist, and n the transducer slope. Parameter n was globally shared, while basal and Em were constrained to 0 and 100%, respectively. Transduction coefficients (τ/KA) were then normalized to the response of endogenous agonist dopamine:

⎛ τ ⎞ ⎛ τ ⎞ ⎛ τ ⎞ Δlog⎜ ⎟ = log⎜ ⎟ − log⎜ ⎟ ⎝ KA ⎠ ⎝ KA ⎠test ligand ⎝ KA ⎠reference ligand Determination of bias was performed by calculation of the difference for two investigated pathways:

⎛ τ ⎞ ⎛ τ ⎞ ⎛ τ ⎞ ΔΔlog⎜ ⎟ = Δlog⎜ ⎟ − Δlog⎜ ⎟ ⎝ KA ⎠1,2 ⎝ KA ⎠signal1 ⎝ KA ⎠signal2 bias factor = 10ΔΔlog(τ / KA) Standard errors were estimated as described previously66 using the following equation:

SEM1,2 =

0.693 k

⎛ ⎞ μL volume of incubation (μL) CL int⎜ ⎟= protein incubation (mg) ⎝ min × mg protein ⎠ 0.693 × T1/2

( ) [X ] + (1 + )

(Em − basal) Y = basal +

n

microsomes (Sprague−Dawley, Sigma-Aldrich, Germany) at a concentration of 0.5 mg microsomal protein/mL in Tris-MgCl2 buffer (48 mM Tris, 4.8 mM MgCl2, pH 7.4). Microsomal reactions were initiated by addition of 50 μL of a cofactor solution (NADPH, final concentration 1 mM, Carl Roth, Germany) and carried out in polyethylene caps (Eppendorf, 1.5 mL) at 37 °C. At 0, 15, 30, and 60 min, the enzymatic reactions were terminated by addition of 500 μL of ice-cold acetonitrile (containing 8 μM internal standard), and precipitated protein was removed by centrifugation (15000g, 3 min). The supernatant was analyzed by HPLC (binary solvent system, eluent methanol in 0.1% aqueous formic acid, 10−90% methanol in 18 min, 90−95% methanol in 2 min, 95% methanol for 2 min, flow rate of 0.5 mL/min) using a Zorbax Eclipse XDB-C8 (4.6 mm × 150 mm, 5 μm) column. For each time point, three independent experiments were performed and parallel control incubations in the absence of cofactor solution were used to determine unspecific binding to the matrix. Substrate remaining was calculated as a mean value ± SEM by comparing the AUC substrate after predetermined incubation time to the AUC of substrate at time 0, corrected by a factor calculated from the AUC of internal standard at each time point. For each compound, the concentration of the substrate remaining was plotted in its logarithmic form as a function of time to calculate an elimination rate constant (k), the half-life (T1/2), and the intrinsic clearance (CLint) according to the following two equations as described previously:54,55

100

current peak,relative = 1+

SEM12 + SEM 2 2

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Statistical significance was calculated using one-way ANOVA followed by Dunnett’s post hoc test at the level of ΔΔlog(τ/KA) and accepting p < 0.05 as level of significance. Metabolic Stability. Metabolic stability of the test compounds was assessed in rat liver microsomes in analogy to previously described procedures.61,68 Each incubation mixture (total volume 500 μL) contained the test compounds (concentration 20 μM from a 10 mM stock solution in DMSO) or a positive control (rotigotine, 20 μM from a 10 mM stock solution in DMSO) and pooled male rat liver

n

( ) [X ] IC50

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00363. Supplementary figures, data from functional assays, 1H and 13C NMR spectra (PDF) 4710



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(10) Urban, J. D.; Clarke, W. P.; von Zastrow, M.; Nichols, D. E.; Kobilka, B.; Weinstein, H.; Javitch, J. A.; Roth, B. L.; Christopoulos, A.; Sexton, P. M.; Miller, K. J.; Spedding, M.; Mailman, R. B. Functional Selectivity and Classical Concepts of Quantitative Pharmacology. J. Pharmacol. Exp. Ther. 2007, 320, 1−13. (11) Neve, K. A. Functional Selectivity of G Protein-Coupled Receptor LigandsNew Opportunities for Drug Discovery. In The Receptors, 1st ed.; Neve, K. A., Ed.; Humana Press: Totowa, NJ; 2009; pp 25−40. (12) Urs, N. M.; Bido, S.; Peterson, S. M.; Daigle, T. L.; Bass, C. E.; Gainetdinov, R. R.; Bezard, E.; Caron, M. G. Targeting B-Arrestin2 in the Treatment of L-DOPA−Induced Dyskinesia in Parkinson’s Disease. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, E2517−E2526. (13) Ahmed, M. R.; Berthet, A.; Bychkov, E.; Porras, G.; Li, Q.; Bioulac, B. H.; Carl, Y. T.; Bloch, B.; Kook, S.; Aubert, I.; Dovero, S.; Doudnikoff, E.; Gurevich, V. V.; Gurevich, E. V.; Bezard, E. Lentiviral Overexpression of GRK6 Alleviates L-DOPA−Induced Dyskinesia in Experimental Parkinson’s Disease. Sci. Transl. Med. 2010, 2, 28ra28. (14) Allen, J. A.; Yost, J. M.; Setola, V.; Chen, X.; Sassano, M. F.; Chen, M.; Peterson, S.; Yadav, P. N.; Huang, X. P.; Feng, B.; Jensen, N. H.; Che, X.; Bai, X.; Frye, S. V.; Wetsel, W. C.; Caron, M. G.; Javitch, J. A.; Roth, B. L.; Jin, J. Discovery of Beta-Arrestin-Biased Dopamine D2 Ligands for Probing Signal Transduction Pathways Essential for Antipsychotic Efficacy. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 18488−18493. (15) Weichert, D.; Kruse, A. C.; Manglik, A.; Hiller, C.; Zhang, C.; Hübner, H.; Kobilka, B. K.; Gmeiner, P. Covalent Agonists for Studying G Protein-Coupled Receptor Activation. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 10744−10748. (16) Ring, A. M.; Manglik, A.; Kruse, A. C.; Enos, M. D.; Weis, W. I.; Garcia, K. C.; Kobilka, B. K. Adrenaline-Activated Structure of Beta2Adrenoceptor Stabilized by an Engineered Nanobody. Nature 2013, 502, 575−579. (17) Rasmussen, S. G. F.; Choi, H.-J.; Fung, J. J.; Pardon, E.; Casarosa, P.; Chae, P. S.; DeVree, B. T.; Rosenbaum, D. M.; Thian, F. S.; Kobilka, T. S.; Schnapp, A.; Konetzki, I.; Sunahara, R. K.; Gellman, S. H.; Pautsch, A.; Steyaert, J.; Weis, W. I.; Kobilka, B. K. Structure of a Nanobody-Stabilized Active State of the B2 Adrenoceptor. Nature 2011, 469, 175−180. (18) Warne, T.; Moukhametzianov, R.; Baker, J. G.; Nehme, R.; Edwards, P. C.; Leslie, A. G. W.; Schertler, G. F. X.; Tate, C. G. The Structural Basis for Agonist and Partial Agonist Action on a B1Adrenergic Receptor. Nature 2011, 469, 241−244. (19) Weichert, D.; Stanek, M.; Hübner, H.; Gmeiner, P. StructureGuided Development of Dual B2 Adrenergic/Dopamine D2 Receptor Agonists. Bioorg. Med. Chem. 2016, 24, 2641−2653. (20) Beattie, D.; Beer, D.; Bradley, M. E.; Bruce, I.; Charlton, S. J.; Cuenoud, B. M.; Fairhurst, R. A.; Farr, D.; Fozard, J. R.; Janus, D.; Rosethorne, E. M.; Sandham, D. A.; Sykes, D. A.; Trifilieff, A.; Turner, K. L.; Wissler, E. An Investigation into the Structure−Activity Relationships Associated with the Systematic Modification of the B2-Adrenoceptor Agonist Indacaterol. Bioorg. Med. Chem. Lett. 2012, 22, 6280−6285. (21) Yoshizaki, S.; Tanimura, K.; Tamada, S.; Yabuuchi, Y.; Nakagawa, K. Sympathomimetic Amines Having a Carbostyril Nucleus. J. Med. Chem. 1976, 19, 1138−1142. (22) Yoshizaki, S.; Manabe, Y.; Tamada, S.; Nakagawa, K.; Tei, S. Isomers of Erythro-5-(1-Hydroxy-2-Isopropylaminobutyl)-8-Hydroxycarbostyril, a New Bronchodilator. J. Med. Chem. 1977, 20, 1103− 1104. (23) Bouyssou, T.; Hoenke, C.; Rudolf, K.; Lustenberger, P.; Pestel, S.; Sieger, P.; Lotz, R.; Heine, C.; Büttner, F. H.; Schnapp, A.; Konetzki, I. Discovery of Olodaterol, a Novel Inhaled B2Adrenoceptor Agonist with a 24 H Bronchodilatory Efficacy. Bioorg. Med. Chem. Lett. 2010, 20, 1410−1414. (24) Hoenke, C.; Bouyssou, T.; Tautermann, C. S.; Rudolf, K.; Schnapp, A.; Konetzki, I. Use of 5-Hydroxy-4H-Benzo[1,4]Oxazin-3Ones as Beta2-Adrenoceptor Agonists. Bioorg. Med. Chem. Lett. 2009, 19, 6640−6644.

Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Author

*Phone: +49 9131 85-29383. Fax: +49 9131 85-22585. E-mail: [email protected]. ORCID

Peter Gmeiner: 0000-0002-4127-197X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grant GRK 1910. We thank Prof. Graeme Milligan (University of Glasgow, U.K.) and Prof. Michel Bouvier (IRIC, University of Montreal) for providing the cDNAs for Gαo1 and GRK2, respectively. We thank Dr. Stefan Löber (FAU Erlangen-Nuernberg) for helpful discussions.

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ABBREVIATIONS USED APPI, atmospheric pressure photoionization; βx -AR, β adrenergic receptor, subtype x; CHO, Chinese hamster ovary; CLint, intrinsic clearance; 1,4-DAP, 1,4-disubstituted-aromatic piperazine; DxR, dopamine Dx receptor; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; Gαx, G protein α subunit subtype x; GRK2, G protein-coupled receptor kinase subtype 2; GTPγS, guanosine 5′-O-(thiotriphosphate); hDxR, human dopamine Dx receptor; h5-HTxR, human serotonin 5-HTx receptor; IKr, rectifying potassium current; L-DOPA, L-3,4-dihydroxyphenylalanine; LID, LDOPA-induced dyskinesia; pα x R, porcine adrenergic α x receptor; p5-HTxR, porcine serotonin 5-HTx receptor; PTX, pertussis toxin; QTc, corrected QT interval; SD, standard deviation; SEM, standard error of mean; TdP, Torsade de Pointes arrhythmia

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REFERENCES

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